Substrates, Peptide Arrays, and Methods

ABSTRACT

Disclosed herein are formulations, substrates, and arrays. Also disclosed herein are methods for manufacturing and using the formulations, substrates, and arrays. Also disclosed are methods for identifying peptide sequences useful for diagnosis and treatment of disorders, and methods for using the peptide sequences for diagnosis and treatment of disorders, e.g., celiac disorder. In certain embodiments, substrates and arrays comprise a porous layer for synthesis and attachment of polymers or biomolecules.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 14/454,554, filed Aug. 7, 2014, which claims the benefit ofInternational Application No. PCT/US2013/025190, filed Feb. 7, 2013,which claims the benefit of the following U.S. Provisional Applications:U.S. Provisional Application No. 61/595,908, filed Feb. 7, 2012, U.S.Provisional Application No. 61/595,988, filed Feb. 7, 2012, U.S.Provisional Application No. 61/608,554, filed Mar. 8, 2012, U.S.Provisional Application No. 61/609,003, filed Mar. 9, 2012, U.S.Provisional Application No. 61/665,489, filed Jun. 28, 2012, U.S.Provisional Application No. 61/726,515, filed Nov. 14, 2012, and U.S.Provisional Application No. 61/761,347, filed Feb. 6, 2013. Thedisclosures of the International Application No. PCT/US2013/02590 andthe above cited U.S. Provisional Applications are incorporated byreference in their entirety for all purposes.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Nov. 13, 2015, is named32561_US_CRF_Sequence_Listing.txt, and is 15,130 bytes in size.

BACKGROUND

A typical microarray system generally comprises biomolecular probes,such as DNA, proteins, or peptides, formatted on a solid planar surfacelike glass, plastic, or silicon chip, plus the instruments needed tohandle samples (automated robotics), to read the reporter molecules(scanners) and analyze the data (bioinformatic tools). Microarraytechnology can facilitate monitoring of many probes per squarecentimeter. Advantages of using multiple probes include, but are notlimited to, speed, adaptability, comprehensiveness and the relativelycheaper cost of high volume manufacturing. The uses of such an arrayinclude, but are not limited to, diagnostic microbiology, including thedetection and identification of pathogens, investigation ofanti-microbial resistance, epidemiological strain typing, investigationof oncogenes, analysis of microbial infections using host genomicexpression, and polymorphism profiles.

Recent advances in genomics have culminated in sequencing of entiregenomes of several organisms, including humans. Genomics alone, however,cannot provide a complete understanding of cellular processes that areinvolved in disease, development, and other biological phenomena;because such processes are often directly mediated by polypeptides.Given that huge numbers of polypeptides are encoded by an organism'sgenome, the development of high throughput technologies for analyzingpolypeptides is of paramount importance.

Peptide arrays with distinct analyte-detecting regions or probes can beassembled on a single substrate by techniques well known to one skilledin the art. A variety of methods are available for creating a peptidemicroarray. These methods include: (a) chemo selective immobilizationmethods; and (b) in situ parallel synthesis methods which can be furtherdivided into (1) SPOT synthesis and (2) photolithographic synthesis.However, the prior art methods suffer from several deficiencies,including limitations on feature density, consistent feature quality(e.g., for step-wise synthesis, coupling efficiencies consistentlyapproaching 98% or higher), and in some aspects, the use of toxicchemicals. The present invention addresses these and other shortcomingsof the prior art, as described below.

SUMMARY

The invention encompasses, in several aspects formulations, substrates,and arrays. The invention also includes methods for manufacturing andusing the formulations, substrates, and arrays.

In one embodiment, the invention includes an array of features attachedto a porous surface layer at positionally-defined locations, thefeatures each comprising: a collection of peptide chains of determinablesequence and intended length, wherein within an individual feature, thefraction of peptide chains within the collection having the intendedlength is characterized by an average coupling efficiency for eachcoupling step of at least 98%.

In certain embodiments, the porous layer comprises a plurality of freecarboxylic acid groups. In one embodiment, the carboxylic acid groupsare oriented in multiple directions. In some embodiments, the porouslayer comprises a plurality of coupling molecules each attached to thearray via a carboxylic acid group. In other embodiments, the porouslayer comprises a plurality of peptide chains each attached to the arrayvia a carboxylic acid group.

In one embodiment, the average coupling efficiency of each coupling stepis at least 98.5%. In some embodiments, the average coupling efficiencyof each coupling step is at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99%. In certain embodiments, each intended length is from 4 to 60 aminoacids in length. In some embodiments, each intended length is at least 5amino acids in length. In one embodiment, each intended length is atleast 6, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 amino acids inlength.

In certain embodiments, each peptide chain comprises one or more L aminoacids. In one embodiment, each peptide chain comprises one or more Damino acids. In certain embodiments, each peptide chain comprises one ormore naturally occurring amino acids. In some embodiments, each peptidechain comprises one or more synthetic amino acids. In some embodiments,the array comprises at least 1,000 different features. In someembodiments, the array comprises at least 10,000 different features.

In certain embodiments, each of the positionally-defined locations is ata different, known location that is physically separated from each ofthe other positionally-defined locations. In some embodiments, each ofthe positionally-defined locations comprises a plurality of identicalsequences. In some embodiments, each positionally-defined locationcomprises a plurality of identical sequences unique from the otherpositionally-defined locations. In some embodiments, each of thepositionally-defined locations is a positionally-distinguishablelocation. In some embodiments, each determinable sequence is a knownsequence. In some embodiments, each determinable sequence is a distinctsequence.

In one embodiment, the features are covalently attached to the surface.In one embodiment, the peptide chains are attached to the porous surfacelayer through a linker molecule or a coupling molecule. In oneembodiment, the features comprise a plurality of distinct, nested,overlapping peptide chains comprising subsequences derived from a sourceprotein having a known sequence. In one embodiment, each peptide chainin the plurality is at least 5 amino acids in length. In someembodiments, each peptide chain in the plurality is at least 5, 10, 15,20, 25, 30, 35, 40, 45, 50, 55, or 60 amino acids in length.

In some embodiments, the features comprise a plurality of peptide chainseach having a random, determinable sequence of amino acids. In someembodiments, the surface comprises any of the substrates describedabove.

In one embodiment, the invention comprises an array of features attachedto a surface at positionally-defined locations, the features eachcomprising: a collection of peptide chains of determinable sequence andintended length, wherein within an individual feature, the fraction ofpeptide chains within the collection having the intended length ischaracterized by an average coupling efficiency for each coupling stepof at least 98%.

In one embodiment, the average coupling efficiency for each couplingstep is at least 98.5%. In one embodiment, the average couplingefficiency for each coupling step is at least 99%. In one embodiment,each intended length is from 4 to 60 amino acids in length. In someembodiments, each intended length is at least 3 amino acids in length.In certain embodiments, each intended length is at least 5 amino acidsin length.

In some embodiments, each intended length is at least 5, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, or 60 amino acids in length. In certainembodiments, each peptide chain comprises one or more L amino acids. Incertain embodiments, each peptide chain comprises one or more D aminoacids. In some embodiments, each peptide chain comprises one or morenaturally occurring amino acids. In one embodiment, each peptide chaincomprises one or more synthetic amino acids.

In one embodiment, the array comprises at least 1,000 differentfeatures. In some embodiments, the array comprises at least 10,000different features.

In certain embodiments, each of the positionally-defined locations is ata different, known location that is physically separated from each ofthe other positionally-defined locations. In some embodiments, each ofthe positionally-defined locations is a positionally-distinguishablelocation. In one embodiment, each determinable sequence is a knownsequence. In one embodiment, each determinable sequence is a distinctsequence. In some embodiments, the features are covalently attached tothe surface. In some embodiments, the peptide chains are attached to thesurface through a linker molecule or a coupling molecule.

In one embodiment, the features comprise a plurality of distinct,nested, overlapping peptide chains comprising subsequences derived froma source protein having a known sequence. In certain embodiments, eachpeptide chain in the plurality is at least 5 amino acids in length.

In one embodiment, each peptide chain in the plurality is at least 5,10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 amino acids in length. Insome embodiments, the features comprise a plurality of peptide chainseach having a random, determinable sequence of amino acids. In someembodiments, the surface comprises the substrate disclosed herein.

In one embodiment, the invention includes a method of producing an arrayof features, comprising: obtaining a surface; and attaching the featuresto the surface, the features each comprising a collection of peptidechains of determinable sequence and intended length, wherein within anindividual feature, the fraction of peptide chains within the collectionhaving the intended length is characterized by an average couplingefficiency for each coupling step of at least 98%.

In one embodiment, the features are attached to the surface using acoupling formulation, comprising a solvent, a water soluble polymer, awater soluble coupling molecule, a water soluble neutralization reagent,and a water soluble coupling reagent.

In one embodiment, the invention includes a method of producing an arrayof features, comprising: obtaining a substrate comprising a planar layercomprising a metal and having an upper surface and a lower surface; anda plurality of pillars operatively coupled to the layer inpositionally-defined locations, wherein each pillar has a planar surfaceextended from the layer, wherein the distance between the surface ofeach pillar and the upper surface of the layer is between 1,000-5,000angstroms, wherein the surface of each pillar is parallel to the uppersurface of the layer, and wherein the plurality of pillars are presentat a density of greater than 10,000/cm²; and coupling through a seriesof coupling reactions the features to the plurality of pillars, thefeatures each comprising a collection of peptide chains of determinablesequence and intended length, wherein within an individual feature, thefraction of peptide chains within the collection having the intendedlength is characterized by an average coupling efficiency for eachcoupling step of at least 98%.

In one embodiment, the features are coupled to the pillars using acoupling formulation, comprising a solvent, a water soluble polymer, awater soluble coupling molecule, a water soluble neutralization reagent,and a water soluble coupling reagent.

In one embodiment, the invention includes a photoactive formulation,comprising: a water soluble photosensitizer, a water soluble photoactive compound, a water soluble polymer, and a solvent.

In certain embodiments, the formulation is selected from the photoactiveformulations shown in Table 1

In some embodiments, the water soluble photosensitizer is athioxanthenone. In some embodiments the water soluble photosensitizer isabout 0.5-5% by weight of the total formulation concentration. In someembodiments, the water soluble photoactive compound comprises aphotoacid generator (PAG) or a photobase generator (PBG). In someembodiments, the photoacid generator is a water soluble iodonium salt, awater soluble polonium salt, or a water soluble sulfonium salt. In someembodiments, the photoacid generator is(4-Methoxyphenyl)phenyliodoniumtrifluoromethanesulfonate. In someembodiments, the photoacid generator is(2,4-dihydroxyphenyl)dimethylsulfonium triflate or (4methoxyphenyl)dimethylsulfonium triflate. In some embodiments, thephotoacid generator is iodonium and sulfonium salts of triflates,phosphates and/or antimonates. In some embodiments, the photoacidgenerator is about 0.5-5% by weight of the total formulationconcentration. In some embodiments, the water soluble polymer is a watersoluble non-crosslinking inert polymer. In some embodiments, the watersoluble polymer is a vinyl pyrrolidone. In some embodiments, the watersoluble polymer is polyvinyl pyrrolidone. In some embodiments, the watersoluble polymer is about 0.5-5% by weight of the total formulationconcentration. In some embodiments, the solvent is water, ethyl lactate,or a combination thereof. In some embodiments, the solvent is about80-90% by weight of the total formulation concentration.

In other embodiments, the invention includes a linker formulation,comprising: a solvent, a water soluble polymer, a water soluble linkermolecule, and a water soluble coupling reagent. In some embodiments, thepolymer is 1% by weight polyvinyl alcohol and 2.5% by weight poly vinylpyrrollidone, the linker molecule is 1.25% by weight polyethylene oxide,the coupling reagent is 1% by weight 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and the solvent is water.

In some embodiments, the solvent is water, an organic solvent, or acombination thereof. In some aspects, the organic solvent is N Methylpyrrolidone, Di methyl formamide, Di chloromethane, Di methyl sulfoxide,or a combination thereof. In some embodiments, the water soluble polymeris a polyvinyl pyrrolidone or a polyvinyl alcohol. In some embodiments,the coupling reagent is a water soluble carbodimide or a water solubletriazole. In some embodiments, the coupling reagent is1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. In some embodiments, thelinker molecule comprises a carboxylic group at a first end of themolecule and a protecting group at a second end of the molecule. In someembodiments, the protecting group is a t-Boc protecting group or anF-Moc protecting group. In some embodiments, the linker molecule is anaryl acetylene, a polyethyleneglycol, a nascent polypeptide, a diamine,a diacid, a peptide, or combinations thereof.

Also encompassed is a coupling formulation, comprising: a solvent, awater soluble polymer, a water soluble coupling molecule, a watersoluble neutralization reagent, and a water soluble coupling reagent.

In some embodiments, the formulation is selected from the coupling filmcontacting formulations shown in Table 2.

In some aspects, the solvent is water, an organic solvent, orcombination thereof. In some embodiments, the organic solvent is NMethyl pyrrolidone, di methyl formamide or combinations thereof. In someembodiments, the polymer is a water soluble vinyl pyrrolidone or a watersoluble vinyl alcohol. In some embodiments, the polymer is 2.5-5% byweight of the total formulation concentration. In some embodiments, theneutralization reagent comprises Hunig's base. In some embodiments, theneutralization reagent is 1-2% by weight of the total formulationconcentration. In some embodiments, the coupling molecule comprises anaturally occurring or artificial amino acid or polypeptide. In someembodiments, the artificial amino acid is a D-amino acid. In someembodiments, the coupling molecule is 1-2% by weight of the totalformulation concentration. In some embodiments, the coupling moleculecomprises a protected side group. In some embodiments, the couplingreagent is water soluble carbodimide or water soluble triazole. In someembodiments, the coupling reagent is 2-4% by weight of the totalformulation concentration.

In some embodiments, the invention includes a substrate, comprising: aplanar layer comprising a metal and having an upper surface and a lowersurface; and a plurality of pillars operatively coupled to the layer inpositionally-defined locations, wherein each pillar has a planar surfaceextended from the layer, wherein the distance between the surface ofeach pillar and the upper surface of the layer is between 1,000-5,000angstroms, wherein the surface of each pillar is parallel to the uppersurface of the layer, and wherein the plurality of pillars are presentat a density of greater than 10,000/cm².

In some embodiments, the surface area of each pillar surface is at least1 μm². In some embodiments, the surface area of each pillar surface hasa total area of less than 10,000 μm². In some embodiments, the distancebetween the surface of each pillar and the lower surface of the layer is2,000-7,000 angstroms. In some embodiments, the layer is 1,000-2,000angstroms thick. In some embodiments, the center of each pillar is atleast 2,000 angstroms from the center of any other pillar. In someembodiments, the metal is chromium. In some embodiments, the metal ischromium, titanium, aluminum, tungsten, gold, silver, tin, lead,thallium, or indium. In some embodiments, the layer is at least 98.5-99%metal. In some embodiments, the layer is a homogenous layer of metal. Insome embodiments, each pillar comprises silicon dioxide or siliconnitride. In some embodiments, each pillar is at least 98-99% silicondioxide.

In some embodiments, the substrate further includes a linker moleculehaving a free amino terminus attached to the surface of each pillar. Insome embodiments, the substrate further includes a linker moleculehaving a free amino terminus attached to the surface of at least onepillar. In some embodiments, the substrate further includes a linkermolecule having a protecting group attached to the surface of eachpillar. In some embodiments, the substrate further includes a linkermolecule having a protecting group attached to the surface of at leastone pillar. In some embodiments, the substrate further includes acoupling molecule attached to the surface of at least one pillar. Insome embodiments, the substrate further includes a coupling moleculeattached to the surface of each pillar. In some embodiments, thesubstrate further includes a water soluble polymer in contact with thesurface of at least one of the pillars. In some embodiments, thesubstrate further includes a water soluble polymer in contact with thesurface of each pillar. In some embodiments, the substrate furtherincludes a gelatinous form of a water soluble polymer in contact withthe surface of at least one of the pillars. In some embodiments, thesubstrate further includes a solid form of a water soluble polymer incontact with the surface of at least one of the pillars.

In some embodiments, the surface of at least one of the pillars isderivatized. In some embodiments, the substrate further includes apolymer chain attached to the surface of at least one of the pillars. Insome embodiments, said polymer chain comprises a peptide chain. In someembodiments, said attachment to the surface of said at least one pillaris via a covalent bond. In some embodiments, the surface of each pillaris square or rectangular in shape. In some embodiments, the substrate iscoupled to a silicon wafer.

In still other embodiments, the invention includes a method of preparinga substrate for attachment of features, comprising: obtaining asubstrate comprising a planar layer comprising a metal and having anupper surface and a lower surface; and a plurality of pillarsoperatively coupled to the layer in positionally-defined locations,wherein each pillar has a planar surface extended from the layer,wherein the distance between the surface of each pillar and the uppersurface of the layer is between 1,000-5,000 angstroms, wherein thesurface of each pillar is parallel to the upper surface of the layer,and wherein the plurality of pillars are present at a density of greaterthan 10,000/cm²; and attaching one or more linker molecules to theplurality of pillars. In some embodiments, the linker molecule isattached using a linker formulation, comprising a solvent, a watersoluble polymer, a water soluble linker molecule, and a water solublecoupling reagent. In some embodiments, the linker molecule comprises aprotecting group.

In some embodiments, the surface of each pillar is parallel to the uppersurface of the layer. In other embodiments, the surface of each pillaris substantially parallel to the upper surface of the layer.

The invention also encompasses a method of preparing a surface forattachment of features, comprising: obtaining a surface and attaching alinker molecule to the surface using a linker formulation, comprising asolvent, a water soluble polymer, a water soluble linker molecule, and awater soluble coupling reagent. In some embodiments, the linker moleculecomprises a protecting group.

In yet other embodiments the invention includes a method of attaching acoupling reagent to a substrate, comprising: obtaining a substratecomprising a planar layer comprising a metal and having an upper surfaceand a lower surface; and a plurality of pillars operatively coupled tothe layer in positionally-defined locations, wherein each pillar has aplanar surface extended from the layer, wherein the distance between thesurface of each pillar and the upper surface of the layer is between1,000-5,000 angstroms, wherein a linker molecule is attached to thesurface of each pillar, and wherein the plurality of pillars are presentat a density of greater than 10,000/cm²; and attaching the couplingreagent to one or more linker molecules. In some embodiments, thecoupling reagent is attached to the one or more linker molecules using acoupling formulation, comprising: a solvent, a water soluble polymer, awater soluble coupling molecule, a water soluble neutralization reagent,and a water soluble coupling reagent. In some embodiments, at least onethe linker molecule is a deprotected linker molecule. In someembodiments, the coupling reagent is an amino acid. In some embodiments,the coupling reagent comprises a protecting molecule.

In some embodiments, the surface of each pillar is parallel to the uppersurface of the layer. In other embodiments, the surface of each pillaris substantially parallel to the upper surface of the layer.

In still other embodiments the invention includes a method of attachinga coupling reagent to a surface, comprising: obtaining a surface havinga linker molecule attached to the surface and attaching the couplingreagent to the linker molecule using a coupling formulation, comprisinga solvent, a water soluble polymer, a water soluble coupling molecule, awater soluble neutralization reagent, and a water soluble couplingreagent. In some aspects, the linker molecule is a deprotected linkermolecule. In some embodiments, the coupling reagent is an amino acid. Insome embodiments, the coupling reagent comprises a protecting molecule.

The invention further includes a method of preparing a substrate forattachment of a coupling reagent, comprising: obtaining a substratecomprising a planar layer comprising a metal and having an upper surfaceand a lower surface; and a plurality of pillars operatively coupled tothe layer in positionally-defined locations, wherein each pillar has aplanar surface extended from the layer, wherein the distance between thesurface of each pillar and the upper surface of the layer is between1,000-5,000 angstroms, wherein a linker molecule is attached to thesurface of each pillar, wherein the substrate is contacted with aphotoactive formulation, and wherein the plurality of pillars arepresent at a density of greater than 10,000/cm²; and applyingultraviolet light to the substrate. In some embodiments, the photoactiveformulation comprises a water soluble photosensitizer, a water solublephoto active compound, a water soluble polymer, and a solvent. In someembodiments, the linker molecule comprises a protecting group. In someembodiments, application of the light to the substrate results inremoval of the protecting group from the linker molecule. In someembodiments, the light is 248 nm light.

In some embodiments, the surface of each pillar is parallel to the uppersurface of the layer. In other embodiments, the surface of each pillaris substantially parallel to the upper surface of the layer.

The invention further includes a method of preparing a surface forattachment of a coupling reagent, comprising: obtaining a surface havinga linker molecule attached to the surface and contacted with aphotoactive formulation comprising a water soluble photosensitizer, awater soluble photo active compound, a water soluble polymer, and asolvent; and applying ultraviolet light to the surface. In someembodiments, the linker molecule comprises a protecting group. In someembodiments, application of the light to the substrate results inremoval of the protecting group from the linker molecule. In someembodiments, the light is 248 nm light.

In still other embodiments the invention includes a method of producinga substrate comprising coupling a planar layer to a plurality ofpillars, wherein the planar layer comprises a metal and has an uppersurface and a lower surface, wherein the plurality of pillars arecoupled to the layer in positionally-defined locations, wherein eachpillar has a planar surface extended from the layer, wherein thedistance between the surface of each pillar and the upper surface of thelayer is between 1,000-5,000 angstroms, and wherein the plurality ofpillars are present at a density of greater than 10,000/cm².

In some embodiments, the surface of each pillar is parallel to the uppersurface of the layer. In other embodiments, the surface of each pillaris substantially parallel to the upper surface of the layer.

In yet other embodiments the invention includes a method of preparing asurface comprising: obtaining a surface comprising silicon dioxide andcontacted with a photoactive formulation comprising a water solublephotosensitizer, a water soluble photo active compound, a water solublepolymer, and a solvent; and applying ultraviolet light topositionally-defined locations located on the top of the surface and incontact with the photoactive formulation, wherein the surface area ofeach positionally-defined location on the surface has a total area ofless than 10,000/μm². In some embodiments, the method further includesremoving the photoactive formulation located external to thepositionally-defined locations. In some embodiments, the method furtherincludes reducing the thickness of the top of the surface locatedexternal to the positionally-defined locations. In some embodiments, themethod further includes depositing a metal layer on the top of thesurface with reduced thickness. In some embodiments, the method furtherincludes removing the photoactive formulation in contact with thepositionally-defined locations located on the top of the surface.

The invention also includes a method of detecting the presence orabsence of a protein of interest in a sample, comprising: obtaining anarray disclosed herein contacted with a sample suspected of comprisingthe protein of interest; and determining whether the protein of interestis present in the sample by detecting the presence or absence of bindingto one or more features of the array.

In still other embodiments the invention includes a method ofidentifying a vaccine candidate, comprising: obtaining an arraydisclosed herein contacted with a sample derived from a subjectpreviously administered the vaccine candidate, wherein the samplecomprises a plurality of antibodies; and determining the bindingspecificity of the plurality of antibodies to one or more features ofthe array. In some embodiments, the features comprise a plurality ofdistinct, nested, overlapping peptide chains comprising subsequencesderived from a source protein having a known sequence.

In some embodiments, the invention comprises a peptide array,comprising: a plurality of peptides coupled to a support; wherein theplurality of peptides comprises overlapping subsequences of a sourceprotein of known sequence. In some embodiments, the source protein isselected from the group consisting of: an alpha gliadin protein, asecalin protein, a hordein protein, a savina protein, a prolaminprotein, or a transglutaminase protein. In certain embodiments, thesource protein is an alpha gliadin protein and wherein the overlappingsubsequences further comprise at least one sequence variant comprising asubstitution of a glutamine residue with a glutamic acid residue.

In some embodiments, the invention comprises a peptide array,comprising: a plurality of peptides coupled to a support; wherein theplurality of peptides comprises a peptide sequence comprising an epitoperecognized by an antibody from a subject diagnosed with celiac disease.

In one embodiment, the plurality of peptides comprises a peptide furthercomprising at least two epitope sequences recognized by an antibody froma subject. In some embodiments, the array is prepared at a density of atleast 10,000 peptide molecules per square centimeter of the substratesurface. In some embodiments, the support comprises a pillar on thesurface of the peptide array. In some embodiments, the peptide arraycomprises a plurality of peptides with a length of 12 or fewer aminoacids.

In some embodiments, the invention comprises a method of diagnosing andisorder in a subject suspected of having the disorder, comprising:obtaining a peptide array, wherein the peptide array comprises a peptidesequence comprising an epitope recognized by an antibody obtained from asubject diagnosed with the disorder; contacting the peptide array with asample obtained from the subject suspected of having the disorder togenerate a signal; and diagnosing the disorder in the subject based onthe signal.

In one embodiment, the disorder is an autoimmune disorder, an infectiousdisease, or a cancer. In some embodiments, the antibody is an autoimmuneantibody. In some embodiments, the antibody is selected from the groupconsisting of: IgG, IgA, IgM, IgD, and IgE.

In certain embodiments, the peptide sequence comprising the epitope iscapable of stimulating an immune response in the subject suspected ofhaving the disorder or in a sample comprising lymphocytes obtained fromthe subject suspected of having the disorder. In some embodiments, theimmune response is measured by an increased quantity of interferon inthe presence of a peptide comprising the epitope. In some embodiments,the epitope is capable of stimulating a B cell from the subject. In someembodiments, the peptide array has a feature density of at least 10,000peptides molecules per square centimeter. In some embodiments, thepeptide array is the array disclosed herein.

In one embodiment, the peptide array comprises a peptide comprising aplurality of epitopes. In certain embodiments, the epitope comprises thesequence: ‘QPEQPF’ (SEQ ID NO: 1). In some embodiments, the method ofdiagnosis has a sensitivity of greater than 99%. In some embodiments,the method of diagnosis has a specificity of greater than 99%. In someembodiments, the method of diagnosis determines subtype of the disorder.In some embodiments, the method of diagnosis determines severity of thedisorder. In certain embodiments, the disorder is celiac disease.

In some embodiments, the invention comprises a method of identifying anepitope sequence associated with a disorder, comprising: providing afirst peptide array disclosed herein; contacting the first peptide arraywith a biological fluid obtained from a subject known to have thedisorder; analyzing the first peptide array to detect binding of anantibody associated with the disorder to at least one peptide sequenceattached to the first peptide array; and identifying an epitope sequencecomprising at least 3 contiguous amino acids by comparing the bindingpattern of antibody to epitope peptide sequences attached to the surfaceof the first peptide array.

In one embodiment, the disorder is an autoimmune disorder, an infectiousdisease, or a cancer. In some embodiments, the biological fluid isselected from the group consisting of: blood, serum, plasma, bile,mucus, pus, or urine. In some embodiments, at least 60% of peptidescomprising the epitope are bound by an antibody associated with thedisorder. In certain embodiments, at least 70% of peptides comprisingthe epitope are bound by an antibody associated with the disorder. Incertain embodiments, at least 80% of peptides comprising the epitope arebound by an antibody associated with the disorder. In certainembodiments, at least 90% of peptides comprising the epitope are boundby an antibody associated with the disorder. In some embodiments, thepercentage of peptides comprising the epitope that are bound to antibodyassociated with the disorder during the first screen is greater when thesample is positive for the disorder than when the sample is negative forthe disorder.

In some embodiments, the invention comprises method of generating apeptide array for diagnosis of an disorder, comprising: providing afirst peptide array disclosed herein; contacting the first peptide arraywith a biological fluid obtained from a subject known to have thedisorder; analyzing the first peptide array to detect binding of anantibody associated with the disorder to at least one peptide sequenceattached to the first peptide array; analytically determining an epitopesequence comprising at least 3 contiguous amino acids from the bindingpattern of antibody to the first peptide array; and generating a peptidearray for diagnosis of the disorder, wherein the peptide array comprisesa peptide comprising the epitope sequence.

In some embodiments, the disorder is an autoimmune disorder, aninfectious disease, or cancer. In certain embodiments, the autoimmunedisorder is celiac disease. In certain embodiments, the biological fluidis selected from the group consisting of: blood, serum, plasma, bile,mucus, pus, or urine.

In some embodiments, the invention comprises an isolated peptidecomprising an epitope identified by the method described herein.

In some embodiments, the invention comprises a method of treating adisorder, comprising administering a composition comprising the isolatedpeptide identified by the method described herein to a subject suspectedof having the disorder. In one embodiment, the disorder is an autoimmunedisease or an infectious disease. In some embodiments, the autoimmunedisorder is celiac disease. In certain embodiments, the peptide is partof a vaccine. In some embodiments, the peptide is administered to thesubject in combination with an adjuvant.

In some embodiments, the invention comprises a peptide array fordiagnosing celiac disease in a suspect suspected of having celiacdisease, comprising: a set of peptides comprising a set of epitopesequences that bind to an antibody associated with celiac disease; and aset of peptide sequences comprising an epitope sequence that binds to aninflammatory response molecule associated with celiac disease. In someembodiments, the peptide array has a feature density of 10,000 peptidesmolecules per square centimeter.

In some embodiments, the invention comprises a substrate, comprising: afirst layer, wherein the layer comprises a plurality of unprotectedcarboxylic acid side groups. In some embodiments, the first layer is aporous layer. In some embodiments, the carboxylic acid side groups areoriented in multiple directions on the surface of the porous layer.

In an embodiment, the first layer is coupled to a support layer. In anembodiment, the first layer is coupled to a silicon wafer. In certainembodiments, the porous layer comprises dextran. In other embodiments,the porous layer comprises porous silica. In an embodiment, the porouslayer comprises pores of a pore size of about 2 nm to 100 μm. In anembodiment, the porous layer comprises a porosity of about 10-80%. In anembodiment, the porous layer comprises a thickness of about 0.01 μm toabout 10,000 μm.

In some embodiments, the substrate further comprises a planar layercomprising a metal having an upper surface and a lower surface. In someembodiments, the first layer is coupled to the planar layer. In someembodiments, the first layer is coated on top of the planar layer. Insome embodiments, the substrate further comprises a plurality of wells.

In an embodiment, the substrate further comprises a plurality of pillarsoperatively coupled to the planar layer in positionally-definedlocations, wherein each pillar has a planar surface extended from theplanar layer, wherein the distance between the surface of each pillarand the upper surface of the layer is between 1,000-5,000 angstroms, andwherein the plurality of pillars are present at a density of greaterthan 10,000/cm², and wherein the first layer is deposited on the planarsurface of the pillars. In some embodiments, the surface area of eachpillar surface is at least 1 μm². In some embodiments, the surface areaof each pillar surface has a total area of less than 10,000 μm². In someembodiments, the distance between the surface of each pillar and thelower surface of the layer is 2,000-7,000 angstroms. In someembodiments, the planar layer is 1,000-2,000 angstroms thick. In someembodiments, the center of each pillar is at least 2,000 angstroms fromthe center of any other pillar. In some embodiments, the surface of eachpillar is parallel to the upper surface of the planar layer. In someembodiments, the surface of each pillar is substantially parallel to theupper surface of the planar layer. In certain embodiments, the metal ischromium. In some embodiments, the metal is chromium, titanium,aluminum, tungsten, gold, silver, tin, lead, thallium, or indium. Insome embodiments, the planar layer is at least 98.5-99% metal by weight.In some embodiments, the planar layer is a homogenous layer of metal. Insome embodiments, each pillar comprises silicon dioxide or siliconnitride. In some embodiments, each pillar is at least 98-99% silicondioxide by weight.

In some embodiments, the substrate comprises a linker molecule having afree amino terminus attached to at least one of the carboxylic acidgroups. In some embodiments, the substrate comprises a linker moleculehaving a free carboxylic acid group attached to at least one of thecarboxylic acid groups. In some embodiments, the substrate comprises acoupling molecule attached to at least one of the carboxylic acidgroups. In some embodiments, the substrate comprises a polymer chainattached to at least one of the carboxylic acid groups. In certainembodiments, the polymer chain comprises a peptide chain. In someembodiments, the polymer chain is attached to at least one of thecarboxylic acid groups via a covalent bond.

In some embodiments, the invention comprises a method for identifying aset of informative peptide sequences, comprising obtaining a datasetcomprising quantitative data indicating specific binding of a ligandpresent in a sample obtained from a subject having a condition to aplurality of peptide sequences; determining, using a specificallyprogrammed computer, a plurality of subsequences present in theplurality of peptide sequences; determining using the specificallyprogrammed computer, the rank number of occurrences of the plurality ofsubsequences in the plurality of peptide sequences; and identifying theset of informative peptide sequences according to the determined rankingof the plurality of subsequences.

In certain embodiments, the method comprises obtaining a datasetcomprising quantitative data indicating specific binding of the ligandpresent in a plurality of samples obtained from subjects having thecondition to a plurality of informative peptides each informativepeptide comprising a plurality of informative peptide sequences;determining using a specifically programmed computer, the fraction ofsamples specifically binding to each of the informative peptides; andidentifying according to the determining a subset of informativepeptides capable of specifically binding to at least 50% of the samples.

In one embodiment, the condition is an autoimmune condition, aninfectious disease condition, or a cancer. In some embodiments, thecondition is an autoimmune condition. In certain embodiments, theautoimmune condition is celiac disease, lupus erythematosis, orrheumatoid arthritis.

In some embodiments, the subset of informative peptides is capable ofspecifically binding to at least 60%, at least 70%, at least 80%, or atleast 90% of the samples.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

These and other features, aspects, and advantages of the presentinvention will become better understood with regard to the followingdescription, and accompanying drawings, where:

FIG. 1 shows a method of manufacturing a substrate.

FIG. 2 shows a method of manufacturing an array.

FIG. 3 diagrams the flow chart of analysis for diagnosing and treatingan autoimmune disorder, according to an embodiment of the invention.

FIG. 4A shows the readout of the fluorescence signal from each of the20-mer experiments.

FIG. 4B shows fluorescence signal intensity vs. each amino acid layer.

FIG. 5 shows the overall step yield analysis via a graph of step yieldvs. each amino acid layer.

FIG. 6 shows the readout of the fluorescence signal from the 1-12-merexperiment.

FIG. 7A shows the readout of the fluorescence signal from a 1-12 mersynthesis of polymeric peptide using standard synthesis-on-a-chipmethods.

FIG. 7B shows the readout of the fluorescence signal from a 1-12 mersynthesis of polyA peptide using standard synthesis-on-a-chip methods.

FIG. 8A shows a comparison of synthesis yield for a 1-12 mer synthesisof heteropolymeric peptides (SEQ ID NOS: 6-13, and 5, respectively, inorder of appearance) using the method described herein vs. the standardchip array synthesis.

FIG. 8B shows a comparison of synthesis yield for a 1-12 mer synthesisof polyA peptides (SEQ ID NOS: 15-22, and 14, respectively, in order ofappearance) using the method described herein vs. the standard chiparray synthesis.

FIG. 8C shows a comparison of coupling efficiency for a 1-12 mersynthesis of heteropolymeric peptides (SEQ ID NOS: 6-13, and 5,respectively, in order of appearance) using the method described hereinvs. the standard chip array synthesis.

FIG. 8D shows a comparison of coupling efficiency for a 1-12 mersynthesis of polyA peptides (SEQ ID NOS: 15-22, and 14, respectively, inorder of appearance) using the method described herein vs. the standardchip array synthesis.

FIG. 9 shows the signal intensity for all twenty amino acids across 12layers.

FIG. 10 shows the normalized signal intensity for all twenty amino acidsgrown at each layer (total of 12 layers) on each wafer.

FIG. 11 shows the expose energy at which acid reaches the substrate forresist combinations. Expose energy is expressed in mJ/cm². Fluorescentsignal intensity is a relative scale from 0 to 65,000.

FIG. 12 shows the expose energy vs. (signal minus noise)/noise (SNR) foreach of the indicated resists and thicknesses.

FIG. 13 shows the expose energy vs. fluorescent signal for each of theindicated resists and thicknesses.

FIG. 14 diagrams the comparison between an antigen-based assay and apeptide array-based assay.

FIG. 15 outlines a method of splitting a protein of known sequence intooverlapping peptide subsequences (SEQ ID NOS: 38-49, respectively, inorder of appearance).

FIG. 16 shows the “deamidation” of peptide sequences (SEQ ID NOS: 46,50, 47, 51, 52, 47, and 53, respectively, in order of appearance) fromalpha gliadin.

FIG. 17 describes a method of determining the immunoactive regions of awhole antigen (Periphilin-1) using a peptide array having subsequences(SEQ ID NOS: 54-59, respectively, in order of appearance) derived fromthe whole antigen.

FIG. 18 shows the binding of an IgA from a celiac positive sample tosequences comprising the epitopes combined according to the matrix.

FIG. 19 shows the binding of an IgG from a celiac positive sample tosequences comprising the epitopes combined according to the matrix.

FIG. 20 depicts process flow for developing wafers in a connectedcoater/developer and litho cell.

FIG. 21 shows the cooling step during wafer production according to theprocess flow.

FIG. 22 depicts coating of a wafer with resist according to the processflow.

FIG. 23 depicts baking of the wafer after coating with resist accordingto the process flow.

FIG. 24 depicts cooling of the wafer after baking according to theprocess flow.

FIG. 25 depicts transfer of the wafers from the coater/developer machineto the scanner.

FIG. 26 depicts baking of the wafer according to the process flow.

FIG. 27 depicts cooling of the wafer after baking according to theprocess flow.

FIG. 28 depicts rinsing of the wafers with deionized water to remove theresist according to the process flow.

FIG. 29 depicts coating of the wafers with amino acid from one of thefirst set of a plurality of reservoirs comprising the selected aminoacid according to the process flow.

FIG. 30 depicts coating of the wafers with amino acid from one of thesecond set of a plurality of reservoirs comprising the selected aminoacid according to the process flow.

FIG. 31 depicts baking of the wafers after addition of the selectedamino acid according to the process flow.

FIG. 32 depicts cooling of the wafer after baking according to theprocess flow.

FIG. 33 depicts coating of the wafers with deionized wafer after coolingaccording to the process flow.

FIG. 34 depicts a step-flow diagram for substrate preparation.

DETAILED DESCRIPTION

Terms used in the claims and specification are defined as set forthbelow unless otherwise specified.

As used herein the term “wafer” refers to a slice of semiconductormaterial, such as silicon or a germanium crystal generally used in thefabrication of integrated circuits. Wafers can be in a variety of sizesfrom, e.g., 25.4 mm (1 inch) to 300 mm (11.8 inches) along one dimensionwith thickness from, e.g., 275 μm to 775 μm.

As used herein the term “photoresist” or “resist” or “photoactivematerial” refers to a light-sensitive material that changes itssolubility in a solution when exposed to ultra violet or deep ultraviolet radiation. Photoresists are organic or inorganic compounds thatare typically divided into two types: positive resists and negativeresists. A positive resist is a type of photoresist in which the portionof the photoresist that is exposed to light becomes soluble to thephotoresist developer. The portion of the photoresist that is unexposedremains insoluble to the photoresist developer. A negative resist is atype of photoresist in which the portion of the photoresist that isexposed to light becomes insoluble to the photoresist developer. Theunexposed portion of the photoresist is dissolved by the photoresistdeveloper.

As used herein the term “photomask” or “reticle” or “mask” refers to anopaque plate with transparent patterns or holes that allow light to passthrough. In a typical exposing process, the pattern on a photomask istransferred onto a photoresist.

As used herein the term “coupling molecule” or “monomer molecule”includes any natural or artificially synthesized amino acid with itsamino group protected with a fluorenylmethyloxycarbonyl group or at-butoxycarbonyl group. These amino acids may have their side chainsprotected as an option. Examples of coupling molecules includeBoc-Gly-Oh, Fmoc-Trp-Oh. Other examples are described below.

As used herein the term “coupling” or “coupling process” or “couplingstep” refers to a process of forming a bond between two or moremolecules such as a linking molecule or a coupling molecule. A bond canbe a covalent bond such as a peptide bond. A peptide bond can be achemical bond formed between two molecules when the carboxyl group ofone coupling molecule reacts with the amino group of the other couplingmolecule, releasing a molecule of water (H₂O). This is a dehydrationsynthesis reaction (also known as a condensation reaction), and usuallyoccurs between amino acids. The resulting CO—NH bond is called a peptidebond, and the resulting molecule is an amide.

As used herein the terms “biomolecule,” “polypeptide,” “peptide,” or“protein” are used interchangeably to describe a chain or polymer ofamino acids that are linked together by bonds. Accordingly, the term“peptide” as used herein includes a dipeptide, tripeptide, oligopeptide,and polypeptide. The term “peptide” is not limited to any particularnumber of amino acids. In some aspects, a peptide contains about 2 toabout 50 amino acids, about 5 to about 40 amino acids, or about 5 toabout 20 amino acids. A molecule, such as a protein or polypeptide,including an enzyme, can be a “native” or “wild-type” molecule, meaningthat it occurs naturally in nature; or it may be a “mutant,” “variant,”“derivative,” or “modification,” meaning that it has been made, altered,derived, or is in some way different or changed from a native moleculeor from another molecule such as a mutant.

As used herein the term “linker molecule” or “spacer molecule” includesany molecule that does not add any functionality to the resultingpeptide but spaces and extends out the peptide from the substrate, thusincreasing the distance between the substrate surface and the growingpeptide. This generally reduces steric hindrance with the substrate forreactions involving the peptide (including uni-molecular foldingreactions and multi-molecular binding reactions) and so improvesperformance of assays measuring one or more aspects of peptidefunctionality.

As used herein the term “developer” refers to a solution that canselectively dissolve the materials that are either exposed or notexposed to light. Typically developers are water-based solutions withminute quantities of a base added. Examples include tetramethyl ammoniumhydroxide in water-based developers. Developers are used for the initialpattern definition where a commercial photoresist is used. Use ofdevelopers is described in Example 1 below.

As used herein the term “protecting group” includes a group that isintroduced into a molecule by chemical modification of a functionalgroup in order to obtain chemoselectivity in a subsequent chemicalreaction. Chemoselectivity refers to directing a chemical reaction alonga desired path to obtain a pre-selected product as compared to another.For example, the use of tboc as a protecting group enableschemoselectivity for peptide synthesis using a light mask and aphotoacid generator to selectively remove the protecting group anddirect pre-determined peptide coupling reactions to occur at locationsdefined by the light mask.

As used herein the term “microarrays” refers to a substrate on whichdifferent probe molecules of protein or specific DNA binding sequenceshave been affixed at separate locations in an ordered manner thusforming a microscopic array.

As used herein the term “microarray system” refers to a system usuallycomprised of biomolecular probes formatted on a solid planar surfacelike glass, plastic or silicon chip plus the instruments needed tohandle samples (automated robotics), to read the reporter molecules(scanners) and analyze the data (bioinformatic tools).

As used herein the term “patterned region” or “pattern” or “location”refers to a region on the substrate on which are grown differentfeatures. These patterns can be defined using photomasks.

As used herein the term “derivatization” refers to the process ofchemically modifying a surface to make it suitable for biomolecularsynthesis. Typically derivatization includes the following steps: makingthe substrate hydrophilic, adding an amino silane group, and attaching alinker molecule.

As used herein the term “capping” or “capping process” or “capping step”refers to the addition of a molecule that prevents the further reactionof the molecule to which it is attached. For example, to prevent thefurther formation of a peptide bond, the amino groups are typicallycapped with an acetic anhydride molecule.

As used herein the term “diffusion” refers to the spread of a chemicalthrough random motion from regions of higher concentration to regions oflower concentration.

As used herein the term “dye molecule” refers to a dye which typicallyis a colored substance that can bind to a substrate. Dye molecules canbe useful in detecting binding between a feature on an array and amolecule of interest.

As used herein, the terms “immunological binding” and “immunologicalbinding properties” refer to the type of non-covalent interactions thatoccurs between an immunoglobulin molecule (or variant thereof such as anscFv) and an antigen for which the immunoglobulin is specific.

As used herein the term “biological sample” refers to a sample derivedfrom biological tissue or fluid that can be assayed for an analyte(s) ofinterest. Such samples include, but are not limited to, sputum, amnioticfluid, blood, blood cells (e.g., white cells), tissue or fine needlebiopsy samples, urine, peritoneal fluid, and pleural fluid, or cellstherefrom. Biological samples may also include sections of tissues suchas frozen sections taken for histological purposes. Although the sampleis typically taken from a human patient, the assays can be used todetect analyte(s) of interest in samples from any organism (e.g.,mammal, bacteria, virus, algae, or yeast) or mammal, such as dogs, cats,sheep, cattle, and pigs. The sample may be pretreated as necessary bydilution in an appropriate buffer solution or concentrated, if desired.

As used herein, the term “assay” refers to a type of biochemical testthat measures the presence or concentration of a substance of interestin solutions that can contain a complex mixture of substances.

The term “subject’ as used herein may refer to a human or any otheranimal having a disorder for testing, diagnosis or treatment.

The term “antigen” as used herein refers to a molecule that triggers animmune response by the immune system of a subject, e.g., the productionof an antibody by the immune system and/or activation of the cellulararm of the immune system (e.g., activation of phagocytes, natural killercells, and antigen-specific cytotoxic T-lymphocytes, along with releaseof various cytokines in response to an antigen). Antigens can beexogenous, endogenous or auto antigens. Exogenous antigens are thosethat have entered the body from outside through inhalation, ingestion orinjection. Endogenous antigens are those that have been generated withinpreviously-normal cells as a result of normal cell metabolism, orbecause of viral or intracellular bacterial infection. Auto antigens arethose that are normal protein or protein complex present in the hostbody but can stimulate an immune response.

As used herein the term “epitope” or “immunoactive regions” refers todistinct molecular surface features of an antigen capable of being boundby component of the adaptive immune system, e.g., an antibody or T cellreceptor. Antigenic molecules can present several surface features thatcan act as points of interaction for specific antibodies. Any suchdistinct molecular feature can constitute an epitope. Therefore,antigens have the potential to be bound by several distinct antibodies,each of which is specific to a particular epitope.

As used herein the term “antibody” or “immunoglobulin molecule” refersto a molecule naturally secreted by a particular type of cells of theimmune system: B cells. There are five different, naturally occurringisotypes of antibodies, namely: IgA, IgM, IgG, IgD, and IgE.

As used herein the term “immune-related molecule” refers to a biologicalmolecule involved in the activation or regulation of an immune response.These include, for example, an antibody, T cell receptor, or MHC complex(e.g., human leukocyte antigen).

As used herein, the term “inflammatory response molecule” refers tomolecules that signal or mediate an inflammatory response, e.g.,cytokines such as interleukin and tumor necrosis factor. Inflammatoryresponse molecules include, for example, pro-inflammatory molecules.

As used herein, the term “autoimmune disorder” refers to any of a largegroup of diseases characterized by abnormal functioning of the immunesystem that causes a subject's immune system to damage the subject's owntissues. Celiac disorder, lupus erythematosis, and rheumatoid arthritisare examples of autoimmune disorders. Autoimmune disorders may beinduced by environmental factors.

The term “percent identity” or “percent sequence identity,” in thecontext of two or more nucleic acid or polypeptide sequences, refer totwo or more sequences or subsequences that have a specified percentageof nucleotides or amino acid residues that are the same, when comparedand aligned for maximum correspondence, as measured using one of thesequence comparison algorithms described below (e.g., BLASTP and BLASTNor other algorithms available to persons of skill) or by visualinspection. Depending on the application, the percent “identity” canexist over a region of the sequence being compared, e.g., over afunctional domain, or, alternatively, exist over the full length of thetwo sequences to be compared.

For sequence comparison, typically one sequence acts as a referencesequence to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyAusubel et al., infra).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J. Mol. Biol. 215:403-410 (1990).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information website.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise.

Compositions

Formulations

Disclosed herein are formulations such as photoactive formulations(e.g., photoresist formulations), coupling formulations, and linkerformulations. These formulations can be useful in the manufacture and/oruse of, e.g., substrates and/or peptide arrays disclosed herein.Generally the components of each formulation disclosed herein aresoluble in water at room temperature (app. 25° C.).

Photoactive Formulations

Disclosed herein are photoactive formulations. In one aspect, aphotoactive formulation can include a chemical amplification resistformulation. In chemical amplification (CA) resists, the primaryphotochemical event produces a mobile catalyst that, typically duringlater postexposure baking (PEB), goes on to induce a cascade of materialtransforming secondary catalytic events within a 5-25 nm radius. Suchchemical amplification thus makes possible an overall quantum yield (thenumber of material reactions divided by number of absorbed photons) ofup to several hundred. A CA resist typically contains a small amount(app. 1-5% by weight) of radiation-sensitive catalyst precursor, e.g., aphotoacid generator (PAG); a plurality of chemical groups that can reactby elimination, addition, or rearrangement in the presence of catalyst;a polymer matrix able to disperse other components in a smooth clearfilm; and optional additives to improve performance or processability,e.g., surfactants, photosensitizers, and etch resistors.

In some aspects, a photoactive formulation is not chemically amplified,i.e., all acid generated is consumed in the reaction (e.g., all the tbocis deprotected and acid is consumed in the reaction). A tboc protectedamino acid can be added along with a photoresist formulation to verifyif chemical amplification occurs. In some aspects, photosensitizers areoptional when 248 nm is used.

In some aspects, a photoactive formulation includes a water solublephotoacid generator and a water soluble photo sensitizer in a polymermatrix dispersed in water. In some aspects, the polymer in thecomposition of the photoresist is generally inert and non-crosslinkingbut the photo reactive components will readily generate sufficientquantities of photoacid upon exposure in a deep ultra violet radiationtool to bring about a desired reaction to produce a product atacceptable yield.

In some aspects, a photoactive formulation can include variouscomponents such as a water soluble photosensitizer, a water solublephoto active compound, a water soluble polymer, and a solvent. Specificexamples of photoactive formulations are shown in Table 1.

Photosensitizers are generally added to a formulation to increase thesensitivity of the photoacid generator and bring the absorption spectrumof the formulation near deep UV (248 nm). In some aspects, a watersoluble photosensitizer can be a thioxanthenone. In some aspects, ageneral thioxanthenone structure is shown below:

In some aspects, the A, R₁, R₂, and R₃ groups of the thioxanthenonestructure shown above can be:

In some aspects, a water soluble photosensitizer can be about 0.5-5% byweight of the total formulation concentration. In some aspects, a watersoluble photosensitizer can be about less than 0.1, 0.1, 0.2, 0.3, 0.4,0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2,3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6,4.7, 4.8, 4.9, 5.0, or greater than 5.0% by weight of the totalformulation concentration.

In some aspects, a water soluble photoactive compound can be a photoacidgenerator (PAG) or a photobase generator (PBG). Photoacid generators (orPAGs) are cationic photoinitiators. A photoinitiator is a compoundespecially added to a formulation to convert absorbed light energy, UVor visible light, into chemical energy in the form of initiatingspecies, e.g., free radicals or cations. Cationic photoinitiators areused extensively in optical lithography. The ability of some types ofcationic photo initiators to serve as latent photochemical sources ofvery strong protonic or Lewis acids is generally the basis for their usein photo imaging applications. In some aspects, a photoacid generator isa water soluble iodonium salt, a water soluble polonium salt, or a watersoluble sulfonium salt. In some aspects, a photoacid generator is(4-Methoxyphenyl)phenyliodonium or trifluoromethanesulfonate. In someaspects, a photoacid generator is (2,4-dihydroxyphenyl)dimethylsulfoniumtriflate or (4 methoxyphenyl)dimethylsulfonium triflate, shown below:

In some aspects, a photoacid generator is iodonium and sulfonium saltsof triflates, phosphates and/or antimonates. In some aspects, aphotoacid generator is about 0.5-5% by weight of the total formulationconcentration. In some aspects, a photoacid generator is about less than0.1, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3,1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7,2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1,4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, or greater than 5.0% byweight of the total formulation concentration.

In some aspects, a water soluble polymer is a water solublenon-crosslinking inert polymer. In some aspects, a water soluble polymeris a polyvinyl pyrrolidone. The general structure of polyvinylpyrrolidone is as follows, where n is any positive integer greater than1:

In some aspects, a water soluble polymer is a polymer of vinylpyrrolidone. In some aspects, a water soluble polymer is polyvinylpyrrolidone. Poly vinyl pyrrollidone is soluble in water and other polarsolvents. When dry it is a light flaky powder, which generally readilyabsorbs up to 40% of its weight in atmospheric water. In solution, ithas excellent wetting properties and readily forms films.

In some aspects, a water soluble polymer is about 0.5-5% by weight ofthe total formulation concentration. In some aspects, a water solublepolymer is about less than 0.1, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2,2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6,3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, orgreater than 5.0% by weight of the total formulation concentration.

In some aspects, a solvent is water, ethyl lactate, or a combinationthereof. In some aspects, ethyl lactate can be dissolved in water tomore than 50% to form a solvent. In some aspects, a solvent can be about10% propylene glycol methyl ether acetate (PGMEA) and about 90% DIwater. In some aspects, a solvent can include up to about 20% PGMEA. Insome aspects, the solvent is about 80-90% by weight of the totalformulation concentration. In some aspects, the solvent is about lessthan 70, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or greater than99% by weight of the total formulation concentration.

In some aspects, a formulation can contain a tboc group that helps inchemical amplification of the initial acid generated upon post exposurebaking. Thus, the formulation can include a tboc protected amino acid,e.g., in order to enhance the chemical amplification duringpost-exposure bake. In some aspects, this tboc protected amino acidwould make up about 0.5-1% by weight of the formulation. In someaspects, a protected amino acid is about 0.5-5% by weight of the totalformulation concentration. In some aspects, a protected amino acid isabout less than 0.1, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0,1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4,2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8,3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, or greaterthan 5.0% by weight of the total formulation concentration.

In any of the combinations above, the formulation can be completelywater strippable even after photo exposure and bake. Thus, in someaspects, only water is used to wash away the photoactive formulationafter exposure and post bake.

Linker Formulations

Also disclosed herein is a linker formulation. A linker formulation caninclude components such as a solvent, a water soluble polymer, a watersoluble linker molecule, and a water soluble coupling reagent. In someaspects, the polymer is 1% by weight polyvinyl alcohol and 2.5% byweight poly vinyl pyrrollidone, the linker molecule is 1.25% by weightpolyethylene oxide, the coupling reagent is 1% by weight1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, and the solvent includeswater. In some aspects, the polymer is 0.5-5% by weight polyvinylalcohol and 0.5-5% by weight poly vinyl pyrrollidone, the linkermolecule is 0.5-5% by weight polyethylene oxide, the coupling reagent is0.5-5% by weight 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, and thesolvent includes water.

In some aspects, the solvent is water, an organic solvent, or acombination thereof. In some aspects, the organic solvent is N Methylpyrrolidone, Di methyl formamide, Di chloromethane, Di methyl sulfoxide,or a combination thereof. In some aspects, the solvent is about 80-90%by weight of the total formulation concentration. In some aspects, thesolvent is about less than 70, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, or greater than 99% by weight of the total formulationconcentration.

In some aspects, a water soluble polymer is a polyvinyl pyrrolidoneand/or a polyvinyl alcohol. The general structure of polyvinyl alcoholis as follows, where n is any positive integer greater than 1:

In some aspects, a water soluble polymer is about 0.5-5% by weight ofthe total formulation concentration. In some aspects, a water solublepolymer is about less than 0.1, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2,2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6,3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, orgreater than 5.0% by weight of the total formulation concentration.

In some aspects, a coupling reagent is a water soluble carbodimide. Insome aspects, a coupling reagent is a water soluble triazole. In someaspects, a coupling reagent is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. In some aspects, a coupling reagent is about 0.5-5% byweight of the total formulation concentration. In some aspects, acoupling reagent is about less than 0.1, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0,2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4,3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8,4.9, 5.0, or greater than 5.0% by weight of the total formulationconcentration.

A linker molecule can be a molecule inserted between a surface disclosedherein and peptide that is being synthesized via a coupling molecule. Alinker molecule does not necessarily convey functionality to theresulting peptide, such as molecular recognition functionality, but caninstead elongate the distance between the surface and the peptide toenhance the exposure of the peptide's functionality region(s) on thesurface. In some aspects, a linker can be about 4 to about 40 atoms longto provide exposure. The linker molecules can be, for example, arylacetylene, ethylene glycol oligomers containing 2-10 monomer units(PEGs), diamines, diacids, amino acids, and combinations thereof.Examples of diamines include ethylene diamine and diamino propane.Alternatively, linkers can be the same molecule type as that beingsynthesized (e.g., nascent polymers or various coupling molecules), suchas polypeptides and polymers of amino acid derivatives such as forexample, amino hexanoic acids. In some aspects, a linker molecule is amolecule having a carboxylic group at a first end of the molecule and aprotecting group at a second end of the molecule. In some aspects, theprotecting group is a t-Boc protecting group or an F-Moc protectinggroup. In some aspects, a linker molecule is or includes an arylacetylene, a polyethyleneglycol, a nascent polypeptide, a diamine, adiacid, a peptide, or combinations thereof. In some aspects, a linkermolecule is about 0.5-5% by weight of the total formulationconcentration. In some aspects, a linker molecule is about less than0.1, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3,1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7,2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1,4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, or greater than 5.0% byweight of the total formulation concentration.

The unbound portion of a linker molecule, or free end of the linkermolecule, can have a reactive functional group which is blocked,protected, or otherwise made unavailable for reaction by a removableprotective group, e.g., t-Boc or F-Moc as noted above. The protectinggroup can be bound to a monomer, a polymer, or a linker molecule toprotect a reactive functionality on the monomer, polymer, or linkermolecule. Protective groups that can be used include all acid and baselabile protecting groups. For example, peptide amine groups can beprotected by t-butoxycarbonyl (t-BOC or BOC) or benzyloxycarbonyl (CBZ),both of which are acid labile, or by 9-fluorenylmethoxycarbonyl (FMOC),which is base labile.

Additional protecting groups that can be used include acid labile groupsfor protecting amino moieties: tert-amyloxycarbonyl,adamantyloxycarbonyl, 1-methylcyclobutyloxycarbonyl,2-(p-biphenyl)propyl(2)oxycarbonyl,2-(p-phenylazophenylyl)propyl(2)oxycarbonyl,alpha,alpha-dimethyl-3,5-dimethyloxybenzyloxy-carbonyl,2-phenylpropyl(2)oxycarbonyl, 4-methyloxybenzyloxycarbonyl,furfuryloxycarbonyl, triphenylmethyl (trityl),p-toluenesulfenylaminocarbonyl, dimethylphosphinothioyl,diphenylphosphinothioyl, 2-benzoyl-1-methylvinyl, o-nitrophenylsulfenyl,and 1-naphthylidene; as base labile groups for protecting aminomoieties: 9 fluorenylmethyloxycarbonyl, methylsulfonylethyloxycarbonyl,and 5-benzisoazolylmethyleneoxycarbonyl; as groups for protecting aminomoieties that are labile when reduced: dithiasuccinoyl, p-toluenesulfonyl, and piperidino-oxycarbonyl; as groups for protecting aminomoieties that are labile when oxidized: (ethylthio)carbonyl; as groupsfor protecting amino moieties that are labile to miscellaneous reagents,the appropriate agent is listed in parenthesis after the group:phthaloyl (hydrazine), trifluoroacetyl (piperidine), and chloroacetyl(2-aminothiophenol); acid labile groups for protecting carboxylic acids:tert-butyl ester; acid labile groups for protecting hydroxyl groups:dimethyltrityl. (See also, Greene, T. W., Protective Groups in OrganicSynthesis, Wiley-Interscience, NY, (1981)).

Coupling Formulations

Also disclosed are coupling formulations. In some aspects, a couplingformulation can include components such as a solvent, a water solublepolymer, a water soluble coupling molecule, a water solubleneutralization reagent, and a water soluble coupling reagent. In someaspects, coupling formulations are shown in Table 2.

In some aspects, a solvent is water, an organic solvent, or combinationthereof. In some aspects, the organic solvent is N Methyl pyrrolidone,di methyl formamide or combinations thereof.

In some aspects, a polymer is a water soluble vinyl pyrrolidone or awater soluble vinyl alcohol. In some aspects, a polymer is 2.5-5% byweight of the total formulation concentration. In some aspects, apolymer is about 0.5-5% by weight of the total formulationconcentration. In some aspects, a polymer is about less than 0.1, 0.1,0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5,1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9,3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3,4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, or greater than 5.0% by weight of thetotal formulation concentration.

In some aspects, a neutralization reagent can include Hunig's base. Thestructure of Hunig's base is:

In some aspects, a neutralization reagent is 1-2% by weight of the totalformulation concentration. In some aspects, a neutralization reagent isabout 0.5-5% by weight of the total formulation concentration. In someaspects, a neutralization reagent is about less than 0.1, 0.1, 0.2, 0.3,0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1,3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5,4.6, 4.7, 4.8, 4.9, 5.0, or greater than 5.0% by weight of the totalformulation concentration.

The coupling molecules can include amino acids. In some instances allpeptides on an array described herein are composed of naturallyoccurring amino acids. In others, peptides on an array described hereincan be composed of a combination of naturally occurring amino acids andnon-naturally occurring amino acids. In other cases, peptides on anarray can be composed solely from non-naturally occurring amino acids.Non-naturally occurring amino acids include peptidomimetics as well asD-amino acids. The R group can be found on a natural amino acid or agroup that is similar in size to a natural amino acid R group.Additionally, unnatural amino acids, such as beta-alanine,phenylglycine, homoarginine, aminobutyric acid, aminohexanoic acid,aminoisobutyric acid, butylglycine, citrulline, cyclohexylalanine,diaminopropionic acid, hydroxyproline, norleucine, norvaline, ornithine,penicillamine, pyroglutamic acid, sarcosine, and thienylalanine can alsobe incorporated. These and other natural and unnatural amino acids areavailable from, for example, EMD Biosciences, Inc., San Diego, Calif. Insome aspects, a coupling molecule comprises a naturally occurring orartificial amino acid or polypeptide. Examples of coupling moleculesinclude Boc-Glycine-OH and Boc-Histine-OH. In some aspects, theartificial amino acid is a D-amino acid. In some aspects, a couplingmolecule is 1-2% by weight of the total formulation concentration. Insome aspects, a coupling molecule is about 0.5-5% by weight of the totalformulation concentration. In some aspects, a coupling molecule is aboutless than 0.1, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1,1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5,2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9,4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, or greater than5.0% by weight of the total formulation concentration. In some aspects,a coupling molecule comprises a protected side group, e.g., a side groupprotected via t-Boc or F-Moc chemistry. In most instances, increasingthe concentration of a coupling molecule provides the best performance.

In some aspects, a coupling reagent is water soluble carbodimide orwater soluble triazole. In some aspects, a coupling reagent is 2-4% byweight of the total formulation concentration. In some aspects, acoupling reagent is about 0.5-5% by weight of the total formulationconcentration. In some aspects, a coupling reagent is about less than0.1, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3,1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7,2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1,4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, or greater than 5.0% byweight of the total formulation concentration.

In any of the combinations above, the formulation can be completelywater strippable.

Substrates

Also disclosed herein are substrates. In some aspects, a substrate caninclude a planar layer comprising a metal and having an upper surfaceand a lower surface; and a plurality of pillars operatively coupled tothe layer in positionally-defined locations, wherein each pillar has aplanar surface extended from the layer, wherein the distance between thesurface of each pillar and the upper surface of the layer is betweenabout 1,000-5,000 angstroms, and wherein the plurality of pillars arepresent at a density of greater than about 10,000/cm². An example of asubstrate is shown in FIG. 1.

In some aspects, the distance between the surface of each pillar and theupper surface of the later can be between about less than 1,000, 2,000,3,000, 3,500, 4,500, 5,000, or greater than 5,000 angstroms (or anyinteger in between).

In some aspects, the surface of each pillar is parallel to the uppersurface of the layer. In some aspects, the surface of each pillar issubstantially parallel to the upper surface of the layer.

In some aspects, the plurality of pillars are present at a density ofgreater than 500, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000,8,000, 9,000, 10,000, 11,000, or 12,000/cm² (or any integer in between).In some aspects, the plurality of pillars are present at a density ofgreater than 10,000/cm². In some aspects, the plurality of pillars arepresent at a density of about 10,000/cm² to about 2.5 million/cm² (orany integer in between). In some aspects, the plurality of pillars arepresent at a density of greater than 2.5 million/cm².

In some aspects, the surface area of each pillar surface is at least 1μm². In some aspects, the surface area of each pillar surface can be atleast 0.1, 0.5, 12, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45,or 50 μm² (or any integer in between). In some aspects, the surface areaof each pillar surface has a total area of less than 10,000 μm². In someaspects, the surface area of each pillar surface has a total area ofless than 500, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000,9,000, 10,000, 11,000, or 12,000 μm² (or any integer in between).

In some aspects, the distance between the surface of each pillar and thelower surface of the layer is 2,000-7,000 angstroms. In some aspects,the distance between the surface of each pillar and the lower surface ofthe layer is about less than 500, 1,000, 2,000, 3,000, 4,000, 5,000,6,000, 7,000, 8,000, 9,000, 10,000, 11,000, 12,000, or greater than12,000 angstroms (or any integer in between). In some aspects, thedistance between the surface of each pillar and the lower surface of thelayer is 7,000, 3,000, 4,000, 5,000, 6,000, or 7,000 angstroms (or anyinteger in between).

In some aspects, the layer is 1,000-2,000 angstroms thick. In someaspects, the layer is about less than 500, 1,000, 2,000, 3,000, 4,000,5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11,000, 12,000, or greaterthan 12,000 angstroms thick (or any integer in between).

In some aspects, the center of each pillar is at least 2,000 angstromsfrom the center of any other pillar. In some aspects, the center of eachpillar is at least about 500, 1,000, 2,000, 3,000, or 4,000 angstroms(or any integer in between) from the center of any other pillar. In someaspects, the center of each pillar is at least about 2 μm to 200 μm fromthe center of any other pillar.

In some aspects, the metal is chromium. In some aspects, the metal ischromium, titanium, aluminum, tungsten, gold, silver, tin, lead,thallium, indium, or a combination thereof. In some aspects, the layeris at least 98.5-99% metal. In some aspects, the layer is 100% metal. Insome aspects, the layer is at least about greater than 90, 91, 92, 93,94, 95, 96, 97, 98, 98.5, or 99% metal. In some aspects, the layer is ahomogenous layer of metal.

In some aspects, at least one or each pillar comprises silicon. In someaspects, at least one or each pillar comprises silicon dioxide orsilicon nitride. In some aspects, at least one or each pillar is atleast 90, 91, 92, 93, 94, 95, 96, 97, 98, 98.5, or 99% silicon dioxide.

In some aspects, a substrate can include a linker molecule having a freeamino terminus attached to the surface of each pillar. In some aspects,a substrate can include a linker molecule having a free amino terminusattached to the surface of at least one pillar. In some aspects, asubstrate can include a linker molecule having a protecting groupattached to the surface of each pillar. In some aspects, a substrate caninclude a linker molecule having a protecting group attached to thesurface of at least one pillar. In some aspects, a substrate can includea coupling molecule attached to the surface of at least one pillar. Insome aspects, a substrate can include a coupling molecule attached tothe surface of each pillar. In some aspects, a substrate can include awater soluble polymer in contact with the surface of at least one ofsaid pillars. In some aspects, a substrate can include a water solublepolymer in contact with the surface of each pillar. In some aspects, asubstrate can include a gelatinous form of a water soluble polymer incontact with the surface of at least one of said pillars. In someaspects, a substrate can include a solid form of a water soluble polymerin contact with the surface of at least one of said pillars.

In some aspects, the surface of at least one of said pillars of thesubstrate is derivatized. In some aspects, a substrate can include apolymer chain attached to the surface of at least one of said pillars.In some aspects, the polymer chain comprises a peptide chain. In someaspects, the attachment to the surface of said at least one pillar isvia a covalent bond.

In some aspects, the surface of each pillar is square or rectangular inshape. In some aspects, the substrate can be coupled to a silicondioxide layer. The silicon dioxide layer can be about 0.5 μm to 3 μmthick. In some aspects, the substrate can be coupled to a wafer, e.g., asilicon wafer. The silicon dioxide layer can be about 700 μm to 750 μmthick.

In some aspects, a substrate can include a porous layer comprisingfunctional groups for binding a first monomer building block.

Porous Layer Substrates

Porous layers which can be used are permeable, polymeric materials ofporous structure which can have a functional group (which is native tothe constituent polymer or which is introduced to the porous layer) forattachment of the first peptide building block. The functional group cancomprise a free carboxylic acid group or a free amino group. Forexample, a porous layer can be comprised of porous silicon withfunctional groups for attachment of a polymer building block attached tothe surface of the porous silicon. In another example, a porous layermay comprise a cross-linked polymeric material. In some embodiments, theporous layer may employ polystyrenes, saccharose, dextrans,polyacryloylmorpholine, polyacrylates, polymethylacrylates,polyacrylamides, polyacrylolpyrrolidone, polyvinylacetates,polyethyleneglycol, agaroses, sepharose, other conventionalchromatography type materials and derivatives and mixtures thereof. Insome embodiments, the porous layer building material is selected from:poly(vinyl alcohol), dextran, sodium alginate, poly(aspartic acid),poly(ethylene glycol), poly(ethylene oxide), poly(vinyl pyrrolidone),poly(acrylic acid), poly(acrylic acid)-sodium salt, poly(acrylamide),poly(N-isopropyl acrylamide), poly(hydroxyethyl acrylate), poly(acrylicacid), poly(sodium styrene sulfonate),poly(2-acrylamido-2-methyl-1-propanesulfonic acid), polysaccharides, andcellulose derivatives. Preferably the porous layer has a porosity of10-80%. In one embodiment, the thickness of the porous layer ranges from0.01 μm to about 1,000 μm. Pore sizes included in the porous layer mayrange from 2 nm to about 100 μm.

According to another aspect of the present invention there is provided asubstrate comprising a porous polymeric material having a porosity from10-80%, wherein reactive groups are chemically bound to the poresurfaces and are adapted in use to interact, e.g. by binding chemically,with a reactive species, e.g., deprotected monomeric building blocks orpolymeric chains. In one embodiment the reactive group is a carboxylicacid group. The carboxylic acid group is free to bind, for example, anunprotected amine group of a peptide or polypeptide. In anotherembodiment, the reactive group is an amino group that is free to bindto, for example, an unprotected carboxylic acid group of a peptide orpolypeptide.

In an embodiment, the porous layer is in contact with a support layer.The support layer comprises, for example, metal, plastic, silicon,silicon oxide, or silicon nitride. In another embodiment, the porouslayer may be in contact with a patterned surface, such as on top ofpillar substrates described above.

Arrays

Also disclosed herein are arrays. In some aspects, an array can be atwo-dimensional array. In some aspects, a two-dimensional array caninclude features attached to a surface at positionally-definedlocations, said features each comprising: a collection of peptide chainsof determinable sequence and intended length, wherein within anindividual feature, the fraction of peptide chains within saidcollection having the intended length is characterized by an averagecoupling efficiency for each coupling step of about 98%. An example ofan array is shown in FIG. 2.

In some aspects, the average coupling efficiency for each coupling stepis at least 98.5%. In some aspects, the average coupling efficiency foreach coupling step is at least 99%. In some aspects, the averagecoupling efficiency for each coupling step is at least 90, 91, 92, 93,94, 95, 96, 97, 98, 98.5, 98.6, 98.7, 98.8, 98.9, 99.0, 99.1, 99.2,99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9, or 100%. In some embodiments,the coupling efficiency is substantially constant over each couplingcycle, and exceeds 98%. In some embodiments the average couplingefficiency exceeds 98% for each coupling step used to synthesize a4-mer, or a 5-mer, or a 6-mer, or a 7-mer or longer polypeptide. In someembodiments the coupling efficiency is substantially constant andexceeds 98% for each coupling step used to synthesize a 4-mer, or a5-mer, or a 6-mer, or a 7-mer or longer polypeptide.

In some aspects, a surface includes a substrate disclosed herein. Insome aspects, a surface is a material or group of materials havingrigidity or semi-rigidity. In some aspects, a surface can besubstantially flat, although in some aspects it can be desirable tophysically separate synthesis regions for different molecules orfeatures with, for example, wells, raised regions, pins, pillars, etchedtrenches, or the like. In certain aspects, a surface may be porous.Surface materials can include, for example, silicon, bio-compatiblepolymers such as, for example poly(methyl methacrylate) (PMMA) andpolydimethylsiloxane (PDMS), glass, SiO₂ (such as, for example, athermal oxide silicon wafer such as that used by the semiconductorindustry), quartz, silicon nitride, functionalized glass, gold,platinum, and aluminum. Functionalized surfaces include for example,amino-functionalized glass, carboxy functionalized glass, and hydroxyfunctionalized glass. Additionally, a surface may optionally be coatedwith one or more layers to provide a second surface for molecularattachment or functionalization, increased or decreased reactivity,binding detection, or other specialized application. Surface materialsand or layer(s) can be porous or non-porous. For example, a surface canbe comprised of porous silicon. Additionally, the surface can be asilicon wafer or chip such as those used in the semiconductor devicefabrication industry. In the case of a wafer or chip, a plurality ofarrays can be synthesized on the wafer.

In some aspects, each peptide chain is from 5 to 60 amino acids inlength. In some aspects, each peptide chain is at least 5 amino acids inlength. In some aspects, each peptide chain is at least 5, 10, 15, 20,25, 30, 35, 40, 45, 50, 55, or 60 amino acids in length. In someaspects, each peptide chain is less than 5, at least 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or greater than60 amino acids in length. In some aspects, each peptide chain comprisesone or more L amino acids. In some aspects, each peptide chain comprisesone or more D amino acids. In some aspects, each peptide chain comprisesone or more naturally occurring amino acids. In some aspects, eachpeptide chain comprises one or more synthetic amino acids.

In some aspects, an array can include at least 1,000 different peptidechains attached to the surface. In some aspects, an array can include atleast 10,000 different peptide chains attached to the surface. In someaspects, an array can include at least 100, 500, 1000, 2000, 3000, 4000,5000, 6000, 7000, 8000, 9000, 10,000, or greater than 10,000 differentpeptide chains attached to the surface (or any integer in between).

In some aspects, each of the positionally-defined locations is at adifferent, known location that is physically separated from each of theother positionally-defined locations. In some aspects, each of thepositionally-defined locations is a positionally-distinguishablelocation. In some aspects, each determinable sequence is a knownsequence. In some aspects, each determinable sequence is a distinctsequence.

In some aspects, the features are covalently attached to the surface. Insome aspects, said peptide chains are attached to the surface through alinker molecule or a coupling molecule.

In some aspects, the features comprise a plurality of distinct, nested,overlapping peptide chains comprising subsequences derived from a sourceprotein having a known sequence. In some aspects, each peptide chain inthe plurality is substantially the same length. In some aspects, eachpeptide chain in the plurality is the same length. In some aspects, eachpeptide chain in the plurality is at least 5 amino acids in length. Insome aspects, each peptide chain in the plurality is at least 5, 10, 15,20, 25, 30, 35, 40, 45, 50, 55, or 60 amino acids in length. In someaspects, each peptide chain in the plurality is less than 5, at least 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,or greater than 60 amino acids in length. In some aspects, at least onepeptide chain in the plurality is at least 5 amino acids in length. Insome aspects, at least one peptide chain in the plurality is at least 5,10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 amino acids in length. Insome aspects, at least one peptide chain in the plurality is less than5, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, or greater than 60 amino acids in length. In someaspects, each polypeptide in a feature is substantially the same length.In some aspects, each polypeptide in a feature is the same length. Insome aspects, the features comprise a plurality of peptide chains eachhaving a random, determinable sequence of amino acids.

Methods

Methods of Manufacturing Substrates

Also disclosed herein are methods for making substrates. In someaspects, a method of producing a substrate can include coupling a planarlayer to a plurality of pillars, wherein the planar layer comprises ametal and has an upper surface and a lower surface, wherein theplurality of pillars are coupled to the layer in positionally-definedlocations, wherein each pillar has a planar surface extended from thelayer, wherein the distance between the surface of each pillar and theupper surface of the layer is between about 1,000-5,000 angstroms, andwherein the plurality of pillars are present at a density of greaterthan about 10,000/cm².

In some aspects, the surface of each pillar is parallel to the uppersurface of the layer. In some aspects, the surface of each pillar issubstantially parallel to the upper surface of the layer.

In some aspects, a method of preparing a substrate surface can includeobtaining a surface comprising silicon dioxide and contacted with aphotoactive formulation comprising a water soluble photosensitizer, awater soluble photo active compound, a water soluble polymer, and asolvent; and applying ultraviolet light to positionally-definedlocations located on the top of the surface and in contact with thephotoactive formulation, wherein the surface area of eachpositionally-defined location on the surface has a total area of lessthan about 10,000/μm². In some aspects, the method can include removingthe photoactive formulation located external to the positionally-definedlocations. In some aspects, the method can include reducing thethickness of the top of the surface located external to thepositionally-defined locations. In some aspects, the method can includedepositing a metal layer on the top of the surface with reducedthickness. In some aspects, the method can include removing thephotoactive formulation in contact with the positionally-definedlocations located on the top of the surface.

In one embodiment, FIGS. 1A-1E presents a process for producing asubstrate.

Referring to FIG. 1A, the first step in the preparation of a substrateis priming a starting wafer in order to promote good adhesion between aphotoactive formulation (e.g., a photoresist) and a surface. Wafercleaning can also be performed, which can include steps such asoxidation, oxide strip, and an ionic clean. Typically deionized (DI)water rinse is used to remove contaminants on the wafer surface. Inwafer fabrication, silane deposition is generally needed to promote thechemical adhesion of an organic compound (photoresist) to a non-organicsubstrate (wafer). The silane acts as a sort of “bridge,” withproperties that will bond to both the photoresist and wafer surface.Typically, hexamethyldisilizane (HMDS) is used. HMDS is an organosiliconcompound that is generally applied on heated substrates in gaseous phasein a spray module or in liquid phase through puddle and spin in adeveloper module followed by a bake step. In a puddle and spin method,HMDS is puddled onto the wafer for a specified time and then spun andbaked at typical temperatures of 110-130° C. for 1-2 mins. In a spraymodule, vapors of HMDS are applied onto a heated wafer substrate at200-220° C. for 30 s-50 s.

Referring to FIG. 1A, after wafer priming, the wafers can be coated witha deep ultra violet (DUV) photoresist in a photoresist coater module.DUV resists are typically polyhydroxystyrene-based polymers with aphotoacid generator providing the solubility change. They can alsocomprise an optional photosensitizer. The matrix in the polymer consistsof a protecting group for e.g., tboc attached to its end group.

The DUV resist is spin coated on the wafers in a photoresist coatmodule. This comprises a vacuum chuck held inside a cup. The wafers aremechanically placed on the chuck by, e.g., a robotic arm and then arespun at required speeds specified by the manufacturer to obtain theoptimum thickness.

Referring to FIG. 1A, the wafers are pre-heated in a pre-heat module.The pre-heat module typically includes a hot plate that can be set torequired temperatures for the corresponding DUV resist as specified bythe manufacturer. The heating can also be done in a microwave for abatch of wafers.

Referring to FIG. 1A, the wafers are now exposed in a deep ultra violetradiation exposure tool through patterned photo masks.

Referring to FIG. 1A, the wafers are now heated in a post exposure bakemodule. This post exposure leads to chemical amplification. The resistmanufacturers provide the typical post exposure bake temperature andtime for their corresponding product. When a wafer coated with a DUVphotoresist is exposed to 248 nm light source through a reticle, aninitial photoacid or photobase is generated. The photoresist is baked topromote diffusion of the photoacid or photobase. The exposed portion ofthe resist becomes soluble to the developer thereby enabling patterningof 0.25 micron dimensions. A post exposure bake module comprises a hotplate set to the required temperatures as specified by the manufacturer.It can consist of three vacuum pins on which the wafers are placed by,e.g., a robotic arm. In other embodiments, the resist process does notuse chemical amplification.

Referring to FIG. 1B, the wafers are now developed in a developermodule. A developer module typically consists of a vacuum chuck that canhold wafers and pressurized nozzles that can dispense the developersolution on to the wafers. The dispense mode can be a puddle and spinmode or a spin and rinse mode. Puddle and spin mode means the wafersremain stationery on the chuck for about 30 sec to 1 minute when thedeveloper solution is dispensed. This puddles the developer solution ontop of the wafer. After a minute, it is spun away. In a spin and rinsemode, the developer solution is dispensed while the wafers are beingspun.

Referring to FIG. 1C, the oxide is now etched away in those regions thatare developed by means of a wet etch or a dry etch process. Etching is aprocess by which material is removed from the silicon substrate or fromthin films on the substrate surface. When a mask layer is used toprotect specific regions of the wafer surface, the goal of etching is toprecisely remove the material that is not covered by the mask. Normally,etching is classified into two types: dry etching and wet etching. Wetetching uses liquid chemicals, primarily acids to etch material, whereasdry etching uses gases in an excited state to etch material. Thesemethods are well known to skilled artisans. These processes can becontrolled to achieve an etch depth of, e.g., 1000 A to 2000 A.

Referring to FIG. 1D, a metal is deposited on the wafers. This metal istypically chromium, titanium, or aluminum. In some embodiments themetals are deposited by a process called sputter deposition. Sputterdeposition is a physical vapor deposition (PVD) method of depositingthin films by sputtering, that is ejecting, material from a “target,”that is a source, which then deposits onto the wafers. The thickness ofmetal deposition is ensured to be at least 500 A on top of thesubstrate, if desired.

Referring to FIG. 1E, the photoresist in between the metal layer and theoxide can be lifted off by using the process diagrammed. In someaspects, the process includes lifting off the resist when the wafer hasa metal layer without affecting the metal layer that previously has beendeposited onto the silicon dioxide. This process results in lift off ofthe photoresist and metal deposited on the top surface of the substratepillars, resulting in a silicon dioxide pillar rising above ametal-coated base that separates adjacent pillars. The wafers aresubmerged in an oxidizer solution overnight and then dipped in a Piranhasolution for typically 1 hr. Piranha solution is a 1:1 mixture ofsulfuric acid and hydrogen peroxide. This can be used to clean all theorganic residues off the substrates. Since the mixture is a strongoxidizer, it will remove most of the organic matter, and it will alsohydroxylate most surfaces (add OH groups), making them hydrophilic. Thisprocess can also include an additional step of plasma ashing.

Surface Derivatization

Substrates can be surface derivatized in a semiconductor module asexplained in U.S. Pat. App. 20100240555, herein incorporated byreference, in its entirety, for all purposes. A typical substrate of thepresent invention has pillars of oxide ready to be surface derivatized.Surface derivatization is a method wherein an amino silane group isadded to the substrate so that free amino groups are available forcoupling the biomolecules. In some aspects, the first molecule to beattached to the surface derivatized substrate is a tboc protectedGlycine. This coupling procedure is similar to a standard Merrifieldsolid phase peptide synthesis procedure which is generally known to oneskilled in this art.

Methods of Manufacturing Arrays

Also disclosed herein are methods for manufacturing arrays. In someaspects, the arrays disclosed herein can be synthesized in situ on asurface, e.g., a substrate disclosed herein. In some instances, thearrays are made using photolithography. For example, masks can be usedto control radiation or light exposure to specific locations on asurface provided with linker molecules having protecting groups. In theexposed locations, the protecting groups are removed, resulting in oneor more newly exposed reactive moieties on the linker. The surface isthen contacted with a solution containing a coupling molecule. Thecoupling molecule can have at least one site that is reactive with thenewly exposed reactive moiety on the linker and at least a secondreactive site protected by one or more protecting groups. The desiredcoupling molecule is then coupled to the unprotected linker molecules.The process can be repeated to synthesize a large number of features inspecific or positionally-defined locations on a surface (see, forexample, U.S. Pat. No. 5,143,854 to Pirrung et al., U.S. PatentApplication Publication Nos. 2007/0154946 (filed on Dec. 29, 2005),2007/0122841 (filed on Nov. 30, 2005), 2007/0122842 (filed on Mar. 30,2006), 2008/0108149 (filed on Oct. 23, 2006), and 2010/0093554 (filed onJun. 2, 2008), each of which is herein incorporated by reference).

In some aspects, a method of producing a two-dimensional array offeatures, can include obtaining a surface; and attaching the features tothe surface, said features each comprising a collection of peptidechains of determinable sequence and intended length, wherein within anindividual feature, the fraction of peptide chains within saidcollection having the intended length is characterized by an averagecoupling efficiency for each coupling step of at least about 98%. Insome aspects, the features are attached to the surface using a couplingformulation, comprising a solvent, a water soluble polymer, a watersoluble coupling molecule, a water soluble neutralization reagent, and awater soluble coupling reagent. In some aspects, the features areattached to the surface using a coupling formulation disclosed herein.In some aspects, the coupling formulation is stripped away using water.

In some aspects, a method of producing a two-dimensional array offeatures, can include obtaining a substrate comprising a planar layercomprising a metal and having an upper surface and a lower surface; anda plurality of pillars operatively coupled to the layer inpositionally-defined locations, wherein each pillar has a planar surfaceextended from the layer, wherein the distance between the surface ofeach pillar and the upper surface of the layer is between about1,000-5,000 angstroms, and wherein the plurality of pillars are presentat a density of greater than about 10,000/cm²; and coupling through aseries of coupling reactions the features to the plurality of pillars,said features each comprising a collection of peptide chains ofdeterminable sequence and intended length, wherein within an individualfeature, the fraction of peptide chains within said collection havingthe intended length is characterized by an average coupling efficiencyfor each coupling step of at least about 98%. In some embodiments, thecoupling efficiency is substantially constant over each coupling cycle,and exceeds 98%. In some embodiments the average coupling efficiencyexceeds 98% for each coupling step used to synthesize a 4-mer, or a5-mer, or a 6-mer, or a 7-mer or longer polypeptide. In some embodimentsthe coupling efficiency is substantially constant and exceeds 98% foreach coupling step used to synthesize a 4-mer, or a 5-mer, or a 6-mer,or a 7-mer or longer polypeptide. Coupling steps used to synthesize. Insome aspects, the features are coupled to the pillars using a couplingformulation, comprising a solvent, a water soluble polymer, a watersoluble coupling molecule, a water soluble neutralization reagent, and awater soluble coupling reagent. In some aspects, the features arecoupled using a coupling formulation disclosed herein. In some aspects,the coupling formulation is stripped away using water. In some aspects,the surface of each pillar is parallel to the upper surface of thelayer. In some aspects, the surface of each pillar is substantiallyparallel to the upper surface of the layer.

In some aspects, a method of preparing a substrate for attachment offeatures, can include obtaining a substrate comprising a planar layercomprising a metal and having an upper surface and a lower surface; anda plurality of pillars operatively coupled to the layer inpositionally-defined locations, wherein each pillar has a planar surfaceextended from the layer, wherein the distance between the surface ofeach pillar and the upper surface of the layer is between about1,000-5,000 angstroms, and wherein the plurality of pillars are presentat a density of greater than about 10,000/cm²; and attaching one or morelinker molecules to the plurality of pillars. In some aspects, thelinker molecule is attached using a linker formulation, comprising asolvent, a water soluble polymer, a water soluble linker molecule, and awater soluble coupling reagent. In some aspects, the linker molecule isattached using a linker formulation disclosed herein. In some aspects,linker molecule comprises a protecting group. In some aspects, thesurface of each pillar is parallel to the upper surface of the layer. Insome aspects, the surface of each pillar is substantially parallel tothe upper surface of the layer.

In some aspects, a method of preparing a surface for attachment offeatures, can include obtaining a surface and attaching a linkermolecule to the surface using a linker formulation, comprising asolvent, a water soluble polymer, a water soluble linker molecule, and awater soluble coupling reagent. In some aspects, linker moleculecomprises a protecting group.

In some aspects, a method of attaching a coupling reagent to asubstrate, can include obtaining a substrate comprising a planar layercomprising a metal and having an upper surface and a lower surface; anda plurality of pillars operatively coupled to the layer inpositionally-defined locations, wherein each pillar has a planar surfaceextended from the layer, wherein the distance between the surface ofeach pillar and the upper surface of the layer is between 1,000-5,000angstroms, wherein a linker molecule is attached to the surface of eachpillar, and wherein the plurality of pillars are present at a density ofgreater than 10,000/cm²; and attaching the coupling reagent to one ormore linker molecules. In some aspects, the coupling reagent is attachedto the one or more linker molecules using a coupling formulation,comprising: a solvent, a water soluble polymer, a water soluble couplingmolecule, a water soluble neutralization reagent, and a water solublecoupling reagent. In some aspects, the coupling reagent is attached tothe one or more linker molecules using a coupling formulation disclosedherein. In some aspects, at least one the linker molecule is adeprotected linker molecule. In some aspects, the coupling reagent is anamino acid. In some aspects, the coupling reagent comprises a protectingmolecule. In some aspects, the coupling formulation is stripped awayusing water. In some aspects, the surface of each pillar is parallel tothe upper surface of the layer. In some aspects, the surface of eachpillar is substantially parallel to the upper surface of the layer.

In some aspects, a method of attaching a coupling reagent to a surfacecan include obtaining a surface having a linker molecule attached to thesurface and attaching the coupling reagent to the linker molecule usinga coupling formulation, comprising a solvent, a water soluble polymer, awater soluble coupling molecule, a water soluble neutralization reagent,and a water soluble coupling reagent. In some aspects, the linkermolecule is a deprotected linker molecule. In some aspects, the couplingreagent is an amino acid. In some aspects, the coupling reagentcomprises a protecting molecule. In some aspects, the couplingformulation is stripped away using water.

In one embodiment, FIGS. 2A through 2G describe a process ofmanufacturing an array.

Referring to FIGS. 2A and 2B, a derivatized surface (e.g., a surfacederivatized wafer) with a linker molecule attached is spun coat with aphotoactive formulation (photoresist) as described herein. The resistthickness can be 100 nm to 200 nm to enable better photoacid diffusion.Hence the spin speed and the speed of spinning can be modified toachieve the desired thickness of the resist.

Referring to FIG. 2B, the resist coat is now baked in a preheat module.The temperatures can be 65° C. to 85° C. for 60 sec to 90 sec. This stepgenerally produces a uniform coat with the photoactive formulation as itinvolves water as a solvent.

Referring to FIG. 2B, the wafer is now exposed in a deep ultra violetscanner tool. The deep UV light is imaged through a photo mask that hasa designed or a random pattern on it. In some aspects, disclosed hereinis a method of selectively irradiating the known features on a substrateor surface to yield photoacid to deprotect the tboc present on the aminoterminal of the linker/coupling molecule. This is possible with designedpatterns on the photo masks that represent the equivalent of one singlemonomer of a known peptide or protein or polypeptide or antigen orantigenic determinant. The expose energy can be from 1 mJ/cm² to 100mJ/cm² in order to produce enough photoacid. A photoacid generated canbe triflic acid which is a super acid with K_(a)=8.0×10¹⁴ mol/kg(pK_(a)˜−15). Triflic acid owes many of its useful properties to itshigh thermal and chemical stability. Owing to its small molecular size,its diffusion is also less burdensome compared to many acids.

Referring to FIG. 2C, the wafer is post baked upon exposure in a postexposure bake module. Post exposure bake acts as a chemicalamplification step. The baking step amplifies the initially generatedphotoacid and also enhances the rate of diffusion to the substrate. Thesubstrate that is already surface derivatized with a linker moleculeattached generally has a protecting group, for example t-boc, on it. Asthe photoacid reaches the substrate, it cleaves the protecting group andhence leaves the amino group of the linker molecule open to be coupledwith the carboxylic acid group of a linker/coupling molecule. The postbake temperature can vary between 75° C. to 115° C., depending on thethickness of the photoresist, for at least 60 sec and not usuallyexceeding 120 sec.

Referring to FIG. 2C, the resist can now be stripped away. In someaspects, provided herein is a method of stripping the photoresistcompletely with DI water. This process is accomplished in a developermodule. The wafer is spun on a vacuum chuck for, e.g., 60 seconds to 90seconds and deionized water is dispensed through a nozzle for about 30seconds.

Referring to FIG. 2D, a coupling formulation is applied on the wafersubstrate. The coupling formulation can be prepared as described hereinor as follows: a solvent, a polymer, a coupling molecule, a neutralizingbase, and one or more coupling reagents. The solvent is generally wateror includes water. A water soluble polymer is first dissolved in knownquantities typically 2.5-5% by weight of the total solutionconcentration. Now the coupling molecule is added at 1-2% by weight ofthe total solution concentration. The neutralizing base is added at thesame concentration as the coupling molecule and the coupling reagents attwice the concentration of the coupling molecule. The addition ofneutralizing base along with the coupling formulation reduces a separatestep in the entire sequence of biomolecular synthesis, as the photoacidleft unreacted on the substrate can be neutralized before coupling. Thisnot only reduces the time to complete a cycle of coupling but alsoimproves the coupling efficiency. The coupling reagents can include awater soluble carbodiimide and a water soluble triazole that, e.g.,prevents racemization of the coupling molecule.

The coupling formulation is applied on the wafer in a coupling spinmodule. A coupling spin module can typically have 20 nozzles or more tofeed the coupling formulation. These nozzles can be made to dispense thecoupling formulation by means of pressurizing the cylinders that holdthese solutions or by a pump that dispenses the required amount. In someaspects, the pump is employed to dispense 5-8 cc of the couplingformulation onto the wafer substrate. The wafer is spun on a vacuumchuck for 15-30 s and the coupling formulation is dispensed. The spinspeed can be set to 2000 to 2500 rpm.

Referring to FIG. 2E, the wafers are now baked in a coupling bakemodule. A coupling bake module is a hot plate set up specifically toreceive wafers just after the coupling formulation is applied. In someaspects, provided herein is a method of baking the spin coated couplingformulation in a hot plate to accelerate the coupling or reactionefficiency. Hot plate baking generally reduces the coupling time foramino acids to less than two minutes with more than 95% coupling orreaction efficiency.

Referring to FIG. 2E, the by-products of the coupling reaction are nowstripped away with DI water in a developer module. The wafer is spun ona vacuum chuck for 60 s to 90 s and DI water is dispensed through anozzle for about 30 secs.

Referring to FIG. 2F, a cap film solution coat is applied on the waferto prevent the unreacted amino groups on the substrate from reactingwith the next coupling molecule. The cap film coat solution can beprepared as follows: a solvent, a polymer, and a coupling molecule. Thesolvent that can be used can be an organic solvent like N methylpyrrolidone, di methyl formamide, or combinations thereof. The cappingmolecule is typically acetic anhydride and the polymer can be polyvinylpyrrolidone, polyvinyl alcohol, polymethyl methacrylate, poly(methyl isopropenyl) ketone, or poly (2 methyl pentene 1 sufone).

This process is done in a capping spin module. A capping spin module caninclude one nozzle that can be made to dispense the cap film coatsolution onto the wafer. This solution can be dispensed throughpressurizing the cylinder that stores the cap film coat solution orthrough a pump that precisely dispenses the required amount. In someaspects, a pump is used to dispense around 5-8 cc of the cap coatsolution onto the wafer substrate. The wafer is spun on a vacuum chuckfor 15-30 s and the coupling formulation is dispensed. The spin speed iscan be set to 2000 to 2500 rpm.

Referring to FIG. 2G, the wafers with the capping solution are baked ina cap bake module. A capping bake module is a hot plate set upspecifically to receive wafers just after the capping film coat isapplied. In some aspects, provided herein is a method of baking the spincoated capping coat solution in a hot plate to accelerate the cappingreaction significantly. Hot plate baking generally reduces the cappingtime for amino acids to less than two minutes.

Referring to FIG. 2G, the byproducts of the capping reaction arestripped in a stripper module. A stripper module can include severalnozzles, typically up to 10, set up to dispense organic solvents such asacetone, iso propyl alcohol, N methyl pyrrolidone, Di methyl formamide,DI water, etc. In some aspects, the nozzles can be designated foracetone followed by iso propyl alcohol to be dispensed onto the spinningwafer. The spin speed is set to be 2000 to 2500 rpm for around 20 s.

This entire cycle of steps from FIG. 2B through 2G can be repeated asdesired with different coupling molecules each time to obtain a desiredsequence.

Methods of Use

Also disclosed herein are methods of using substrates, formulations,and/or arrays. Uses of the arrays disclosed herein can include researchapplications, therapeutic purposes, medical diagnostics, and/orstratifying one or more patients or subjects.

Any of the arrays described herein can be used as a research tool or ina research application. In one aspect, arrays can be used for highthroughput screening assays. For example, enzyme substrates (i.e.,peptides on a peptide array described herein) can be tested bysubjecting the array to an enzyme and identifying the presence orabsence of enzyme substrate(s) on the array, e.g., by detecting at leastone change among the features of the array.

Arrays can also be used in screening assays for ligand binding, todetermine substrate specificity, or for the identification of peptidesthat inhibit or activate proteins. Labeling techniques, protease assays,as well as binding assays useful for carrying out these methodologiesare generally well-known to one of skill in the art.

In some aspects, an array can be used to represent a known proteinsequence as a sequence of overlapping peptides. For example, the aminoacid sequence of a known protein is divided into overlapping sequencesegments of any length and of any suitable overlapping frame, andpeptides corresponding to the respective sequence segments are in-situsynthesized as disclosed herein. The individual peptide segments sosynthesized can be arranged starting from the amino terminus of theknown protein.

In some aspects, an array is used in a method wherein the antigenicrepresentation of the array includes at least one region where the wholeantigen sequence of a known protein is spanned via epitope sliding; theimmunoactive regions of the antigen are determined by contacting one ormore clinical samples on the array or a plurality of different arrays,and the set of peptide sequences required to represent the known proteinantigen are reduced.

In some aspects, a sample is applied to an array having a plurality ofrandom peptides. The random peptides can be screened and BLASTed todetermine homologous domains with, e.g., a 90% or more identity to agiven antigenic sequence. In some aspect, the whole antigenic sequencecan then be synthesized and used to identify potential markers and/orcauses of a disease of interest.

In some aspects, an array is used for high throughput screening of oneor more genetic factors. Proteins associated with a gene can be apotential antigen and antibodies against these gene related proteins canbe used to estimate the relation between gene and a disease.

In another example, an array can be used to identify one or morebiomarkers. Biomarkers can be used for the diagnosis, prognosis,treatment, and management of diseases. Biomarkers may be expressed, orabsent, or at a different level in an individual, depending on thedisease condition, stage of the disease, and response to diseasetreatment. Biomarkers can be, e.g., DNA, RNA, proteins (e.g., enzymessuch as kinases), sugars, salts, fats, lipids, or ions.

Arrays can also be used for therapeutic purposes, e.g., identifying oneor more bioactive agents. A method for identifying a bioactive agent cancomprise applying a plurality of test compounds to an array andidentifying at least one test compound as a bioactive agent. The testcompounds can be small molecules, aptamers, oligonucleotides, chemicals,natural extracts, peptides, proteins, fragment of antibodies, antibodylike molecules or antibodies. The bioactive agent can be a therapeuticagent or modifier of therapeutic targets. Therapeutic targets caninclude phosphatases, proteases, ligases, signal transduction molecules,transcription factors, protein transporters, protein sorters, cellsurface receptors, secreted factors, and cytoskeleton proteins.

In another aspect, an array can be used to identify drug candidates fortherapeutic use. For example, when one or more epitopes for specificantibodies are determined by an assay (e.g., a binding assay such as anELISA), the epitopes can be used to develop a drug (e.g., a monoclonalneutralizing antibody) to target antibodies in disease.

In one aspect, also provided are arrays for use in medical diagnostics.An array can be used to determine a response to administration of drugsor vaccines. For example, an individual's response to a vaccine can bedetermined by detecting the antibody level of the individual by using anarray with peptides representing epitopes recognized by the antibodiesproduced by the induced immune response. Another diagnostic use is totest an individual for the presence of biomarkers, wherein samples aretaken from a subject and the sample is tested for the presence of one ormore biomarkers.

Arrays can also be used to stratify patient populations based upon thepresence or absence of a biomarker that indicates the likelihood asubject will respond to a therapeutic treatment. The arrays can be usedto identify known biomarkers to determine the appropriate treatmentgroup. For example, a sample from a subject with a condition can beapplied to an array. Binding to the array may indicate the presence of abiomarker for a condition. Previous studies may indicate that thebiomarker is associated with a positive outcome following a treatment,whereas absence of the biomarker is associated with a negative orneutral outcome following a treatment. Because the patient has thebiomarker, a health care professional may stratify the patient into agroup that receives the treatment.

In some aspects, a method of detecting the presence or absence of aprotein of interest in a sample can include obtaining an array disclosedherein and contacted with a sample suspected of comprising the proteinof interest; and determining whether the protein of interest is presentin the sample by detecting the presence or absence of binding to one ormore features of the array.

In some aspects, a method of identifying a vaccine candidate can includeobtaining an array disclosed herein contacted with a sample derived froma subject previously administered the vaccine candidate, wherein thesample comprises a plurality of antibodies; and determining the bindingspecificity of the plurality of antibodies to one or more features ofthe array. In some aspects, the features comprise a plurality ofdistinct, nested, overlapping peptide chains comprising subsequencesderived from a source protein having a known sequence.

In one embodiment, a method of diagnosing and treating an autoimmunedisorder is provided. In one embodiment, use of the peptide chip todetecting multiplex antibodies in a serum sample is provided. In someaspects, this method is performed in a single assay. In some aspects,this method is performed on a single peptide chip. In one embodiment,this method provides the ability to detect multiple chemokines from anautoimmune disorder. In one embodiment, this method provides the abilityto identify the subtype and severity of an autoimmune disorder.

In one embodiment, methods of diagnosing using the peptide chip have areproducibility of R² greater than 0.95. In some embodiments, themethods of diagnosing an autoimmune disorder using the peptide chip havea specificity of greater than 0.99 and/or a sensitivity of greater than0.99.

In one embodiment, the autoimmune disorder is celiac disease. In anotherembodiment, the autoimmune disorder is lupus erythematosis. In anotherembodiment, the autoimmune disorder is rheumatoid arthritis.

The peptide array disclosed herein may be used to identify epitopesrelated to autoimmune diseases. In one embodiment, the epitopes are Bcell epitopes, T cell epitopes, or epitopes related to inflammatoryresponse (e.g., TNF). Epitopes related to inflammatory response may beidentified by the present invention using a cytokine assay. In oneembodiment, the peptide sequences identified by this cytokine assay maybe used in immunosuppressive vaccines. In other embodiments, the peptidesequences may be used as part of a peptide array to identify thepresence of inflammatory molecules in a subject suspected of having aninflammatory disorder, e.g., an autoimmune disorder. In one embodiment,the peptide array may be used to identify B cell epitopes. In thisembodiment, epitopes binding to antibodies from a sample associated withan autoimmune disorder are identified. These peptides are then used onanother peptide array useful for diagnosis of an autoimmune disorder. Inone embodiment, diagnosis of an autoimmune disorder includesidentification of autoimmune disorder subtype. In some embodiments, theidentified B cell epitopes are used to measure a patient's response totreatment of an autoimmune disorder. In one embodiment, T cell epitopesmay be identified by the present invention using an MHC complex assay(e.g., a human leukocyte antigen assay). Epitopes identified asinteracting with the MHC complex in a subject identified as having anautoimmune disorder may be used for treatment of the autoimmunedisorder. Such peptides may be useful in a vaccine or other drugs for Tcell regulation. A flow chart depicting the identification of epitopesequences and their use, according to several embodiments of theinvention, is shown in FIG. 3.

In some aspects the invention includes bioinformatic analysis of datato, e.g., identify informative sub-sequences, and subsequent synthesisand testing of synthetic peptide sequences useful for diagnosing acondition. These bioinformatic methods are carried out, in part, using acomputer to accomplish one or more of the following steps: 1) generatingsubsequences from longer sequences; 2) tabulating and ranking theoccurrence of subsequences in positive hits from samples bound to arraysof tiled naturally-occurring peptide sequences; 3) analyzing hits toarrays comprising synthetic sequences that include informativesubsequences.

Unless specifically stated otherwise as apparent from the followingdiscussion, it is appreciated that throughout the description,discussions utilizing terms such as “processing” or “computing” or“calculating” or “determining” or “displaying” or “analyzing” or“comparing” or “identifying” or the like, refer to the action andprocesses of a computer system, or similar electronic computing device,that manipulates and transforms data represented as physical(electronic) quantities within the computer system's registers andmemories into other data similarly represented as physical (electronic)quantities within the computer system's registers and memories intoother data similarly represented as physical quantities within thecomputer system memories or registers or other such information storage,transmission or display devices.

The present invention also relates to system apparatus for performingthe operations herein. This apparatus may be specially constructed forthe required purposes, or it may comprise a general purpose computerselectively activated or reconfigured by a computer program stored inthe computer. Such a computer program may be stored in a computerreadable storage medium, such as, but is not limited to, any type ofdisk including floppy disks, optical disks, CD-ROMs, andmagnetic-optical disks, read-only memories (ROMs), random accessmemories (RAMs), EPROMs, EEPROMs, magnetic or optical cards, or any typeof media suitable for storing electronic instructions, and each coupledto a computer system bus.

Various general purpose systems may be used with programs in accordancewith the teachings herein, or it may prove convenient to construct morespecialized apparatus to perform the required method procedures. Therequired structure for a variety of these systems will appear from thedescription below. In addition, the present invention is not describedwith reference to any particular programming language. It will beappreciated that a variety of programming languages may be used toimplement the teachings of the invention as described herein.

EXAMPLES

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of protein chemistry, biochemistry,recombinant DNA techniques and pharmacology, within the skill of theart. Such techniques are explained fully in the literature. See, e.g.,T. E. Creighton, Proteins: Structures and Molecular Properties (W.H.Freeman and Company, 1993); A. L. Lehninger, Biochemistry (WorthPublishers, Inc., current addition); Sambrook, et al., MolecularCloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology(S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington'sPharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack PublishingCompany, 1990); Carey and Sundberg Advanced Organic Chemistry 3^(rd) Ed.(Plenum Press) Vols A and B (1992).

Example 1 Substrate Production

This example describes construction of a substrate. This process isvisually outlined in FIG. 1. Silicon wafers with 2.4 μm thermally grownoxide were obtained from University Wafers. These wafers were firstprimed with a primer in a spray module. Hexamethyl disilazane (HMDS) wasobtained from Sigma Aldrich Inc. The wafers were then spun coat in aphotoresist coat module with a commercially available deep Ultra violetphotoresist, P5107 obtained from Rohm and Haas or AZ DX7260p 700 from AZElectronic Materials, to obtain a thickness of 6000 Å. The wafers werethen baked in a hot plate at 120° C. for 60 seconds.

Photomasks that have the patterned regions to create the features wereused to image the array on to the substrate surface. The wafers werethen exposed in a 248 nm deep ultra violet radiation scanner tool, Nikon5203, with expose energy of 18 mJ/cm2. The wafers were then postexposure baked at 110° C. for 120 seconds in a hot plate and developedwith commercially available NMD-3 developer, obtained from Tokyo OhkaKogyo Co., Ltd., for 60 seconds.

After this the oxide was etched by using either a wet etch process ordry plasma etch process. Standard semiconductor etch techniques wereused. Oxide etch depths were from 1000 Å to 2000 Å.

After etching, chromium was deposited to a thickness of 500 Å to 1500 Åby a physical deposition method. Standard etching and metal depositiontechniques were employed.

After the chromium was deposited, the resist was lifted off with thefollowing process: The wafer was left in Nanostrip obtained from CyantekInc. overnight and then dipped in Piranha solution for 90 mins. Piranhasolution is a 50:50 mixture of sulfuric acid and hydrogen peroxide.Sulfuric acid and hydrogen peroxide were obtained from Sigma AldrichCorp. Plasma ashing was performed to oxidize the remaining impurities.This process produced a substrate having pillars of silicon dioxideseparated by metal.

Alternatively, the deposited chromium was also polished to a depth of500 Å to 1500 A, depending on the deposition. The polishing wasperformed to obtain pillars of silicon dioxide separated by metal. Theseparation of each pillar from center to center was 70,000 Å. Thesurface area of top of each pillar was 3,500 Å×3,500 Å.

Derivatization: The wafers were then surface derivatized using thefollowing method: Aminopropyl triethoxy silane (APTES) was obtained fromSigma Aldrich. Ethanol 200 proof was obtained from VWR. The wafers werefirst washed with ethanol for 5 minutes and then in 1% by weightAPTES/Ethanol for 20-30 minutes to grow the silane layer. Then thewafers were cured in a 110° C. nitrogen bake oven to grow a mono silanelayer with a —NH₂ group to attach a linker molecule.

Example 2 Photoresist Formulation Production and Use

Water Soluble Photoresists were Prepared as Follows:

The water soluble compounds were obtained from Hampford Research Inc.These included a water soluble thioxanthenone derivative and a watersoluble photoacid generator (PAG). Polyvinylpyrrollidone (PVP) wasobtained from Polysciences Inc.

2% by weight (0.5%-5% by weight was tested and gave similar results;data not shown) of PVP was dissolved in water. Next the photoacidgenerator (4% by weight) and the photo initiators (4% by weight) wereadded at a ratio of 1:2 with respect to PVP. All of the components werethen left spinning in a magnetic stirrer overnight to obtain the finalcomposition. The inclusion of photoinitiators is optional.

Specific photoactive formulations produced using the above procedures(as a general guide) are shown in Table 1. Each formulation from Table 1was made by selecting a single cell from each column within a particularformulation row (1-4). For instance, one formulation (from formulationrow 1 from Table 1, top row) included the following components:Polyethylene glycol monomethyl ether 2 wt %; Polyvinyl pyrrollidone 2 wt%; 4 Methoxyphenyl)phenyliodonium trifluoromethanesulfonate 5 wt %;Water soluble Isopropyl thioxanthenone (ITX) 5 wt % (Lin et al.,“Synthesis of Water Soluble Photoinitiators of Thioxanthone DerivativesIII” Huadong Ligong Daxue Xuebao, vol. 26, No. 2, 2000, pp. 212-214,220); and the remainder of the formulation being solvent (the solventbeing 100% water). In another instance, a different formulation (fromformulation row 2 from Table 1, top row) included the followingcomponents: Polyethylene glycol monomethyl ether 2 wt %; Polyvinylpyrrollidone 2 wt %; (4 methoxyphenyl)dimethylsulfonium triflate 5 wt %;Water soluble ITX 5 wt %; and the remainder of the formulation beingsolvent (the solvent being 90% by weight water and 10% by weightPropylene glycol methyl ether acetate (PGMEA)).

The formulations were prepared as described above. Each photoresistformulation was then spin coated on a substrate (see above) at varyingspeeds to obtain the desired thickness.

TABLE 1 Photoactive Formulations Formulation Polymer Photoacidgenerators Photo initiators Solvent (wt %) 1 Polyethylene glycol 4Methoxyphenyl)phenyliodonium Water soluble Water 100% monomethyl ether 2wt % trifluoromethanesulfonate 5 wt % ITX 5 wt % Polyvinyl pyrrollidone2 wt % Poly (2- dimethylaminoethyl methacrylate) 2.5 wt % Poly(2-hydroxypropyl methacrylate) 2.5 wt % Poly 4 vinyl pyridine 5 wt % 2Polyethylene glycol (4 Water soluble Water 90% monomethyl ether 2 wt %methoxyphenyl)dimethylsulfonium ITX 5 wt % PGMEA 10% Polyvinylpyrrollidone triflate 5 wt % 2 wt % Poly (2- dimethylaminoethylmethacrylate) 2.5 wt % Poly (2-hydroxypropyl methacrylate) 2.5 wt % Poly4 vinyl pyridine 5 wt % 3 Polyethylene glycol (2,4- Water soluble Water90% monomethyl ether 2 wt % dihydroxyphenyl)dimethylsulfonium ITX 5 wt %ethyl lactate Polyvinyl pyrrollidone triflate 2.5 wt % 10% 2 wt % Poly(2- (4 dimethylaminoethyl methoxyphenyl)dimethylsulfonium methacrylate)2.5 wt % triflate 2.5 wt % Poly (2-hydroxypropyl methacrylate) 2.5 wt %Poly 4 vinyl pyridine 5 wt % 4 Polyethylene glycol (2,4- Water solubleWater 50% monomethyl ether 2 wt % dihydroxyphenyl)dimethylsulfonium ITX5 wt % ethyl lactate Polyvinyl pyrrollidone triflate 2.5 wt % 50% 2 wt %Poly (2- (4 dimethylaminoethyl methoxyphenyl)dimethylsulfoniummethacrylate) 2.5 wt % triflate 2.5 wt % Poly (2-hydroxypropylmethacrylate) 2.5 wt % Poly 4 vinyl pyridine 5 wt % Note: The % in thesolvent column refers to the contents of the solvent itself. E.g., 100%water means that the solvent is 100% water.

Example 3 Coupling Formulation and Capping Solution Production and Use

Water-Based Coupling Solutions for the 20 Natural Amino Acids werePrepared as Follows:

Water soluble inert polymers such as poly vinyl alcohol (PVA), polyvinylpyrrolidone (PVP), or polyethyleneglycol were obtained from PolysciencesInc.

EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide), and HonB(n-hydroxy-5-norbornene-2,3-dicarboximide) were obtained from SigmaAldrich Corp. DIEA (Diisopropylethylamine) was obtained from SigmaAldrich Corp.

All t-boc/Fmoc protected amino acids were obtained from AAPPTEC/Anaspec.The inert water soluble polymers 3% by weight PVP and 7% by weight PVAwere dissolved in the ratio of 1:2.5 in deionized (DI) water which makesup about 10% by weight of the solution along with 2% by weight aminoacid concentration. 4% by weight for each EDC and HonB were added asreagents twice the concentration of amino acids. DIEA (4% by weight) wasadded at the same concentration as EDC and HoNb. This water couplingsolution was spin coated on a derivatized substrate to form a uniformsolid layer all available to couple to the substrate. The wafer was thenbaked on a hot plate for 2 minutes at 90° C. to remove the remainingsolvent (DI water) and coupled at the same time. Next the coupling coatwas washed away with DI water in a strip module.

Specific coupling formulations produced using the above procedures (as ageneral guide) are shown in Table 2. Each formulation from Table 2 wasmade by selecting a single cell from each column within a particularformulation row (1-20). For instance, one formulation (from formulationrow 1 from Table 2, top row) included the following components:Polyvinyl alcohol: 10% by weight; Boc-Ala-OH, Boc-Ala-NH₂, Boc-Ala-Osu(Osu=Oxy succinimide) (Concentration—0.2M and 0.3M);1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (Concentration-0.3M); 50%N-methylpyrrolidone+50% Diisopropylethylamine (total concentration0.3M); and the remainder of the formulation being solvent (the solventbeing 100% water). In another instance, a different formulation (fromformulation row 2 from Table 2, top row) included the followingcomponents: Polyvinyl alcohol-10% by weight; Boc-Arg-OH,Boc-Arg-(Mts)-OH (Mts=Mesitylene sulfonyl), Boc-Arg(Z)-OH(Concentration—0.2M and 0.3M); 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (Concentration-0.3M); 50% N-methylpyrrolidone+50%Diisopropylethylamine (total concentration 0.3M); and the remainder ofthe formulation being solvent (the solvent being 100% water).Formulations for each of the 20 standard amino acids are shown in Table2. Each coupling formulation comprises at least one polymer, amino acid,coupling reagent, neutralizing agent, and solvent selected from therespective columns for each formulation in Table 2.

The formulations were prepared as described above.

Capping Solution was Prepared as Follows:

Acetic anhydride was obtained from Sigma Aldrich Corp. PVP was dissolvedin N-methylpyrrolidone which makes up 2% by weight (1-2% by weight wastested and gave similar results; data not shown) of the total solution.Next acetic anhydride was added to make up 25% by weight (20-30% byweight was tested and gave similar results; data not shown) of thesolution. This capping solution was then spin coated on the wafers in acapping module by spinning the wafers at 2000 rpm for 30 seconds. Thewafers were then baked in a cap bake module for up to 2 minutes at 75°C. to complete the capping process. The remaining solution was washedaway with DI water in a strip module.

The above processes are visually outlined in FIG. 2.

TABLE 2 Coupling Formulations Formulation Polymer Amino acid Couplingreagent Neutralizing agent Solvent (wt %) 1 Polyvinyl alcohol- Boc-Ala-OH, Boc-Ala- 1-ethyl-3-(3- 50 wt % N methyl 100% Water 10 wt % NH2,Boc- Als-Osu dimethylaminopropyl) Pyrrolidone + 50 wt % PolyvinylConcentration- 0.2M and carbodiimide) Di isopropyl ethyl 50% waterpyrrolidone-10 wt % 0.3M Concentration-0.3M amine 50% N Totalconcentration Methyl 0.3M pyrrollidone Polyvinyl alcohol n hydroxy 5norbornene 90% water 2.5 wt % Polyvinyl 2,3 di carboximide 10% Npyrrolidone 2.5 wt % Concentration-0.2M Methyl pyrrollidone Polyethylene glycol 80% water 5 wt % Poly 2 vinyl 20% N pyridine N oxideMethyl 5 wt % pyrrollidone Poly ethylene glycol 10% dimethyl monomethylether formamide 10 wt % Polyethylene glycol 80% water monomethylether20% N 5 wt % Poly vinyl Methyl alcohol 2.5 wt % pyrrollidone Poly vinylpyrrolidone 2.5 wt % Polyethylene glycol dimethylether 5 wt % Poly vinylalcohol 2.5 wt % Poly vinyl pyrrolidone 2.5 wt % 2 Polyvinyl alcohol-Boc- Arg-OH, Boc-Arg 1-ethyl-3-(3- 50 wt % N methyl 100% Water 10 wt %(Mts)-OH, Boc- Arg(Z)- dimethylaminopropyl) Pyrrolidone + 50 wt %Polyvinyl OH carbodiimide) Di isopropyl ethyl 50% water pyrrolidone-10wt % Concentration- 0.2M and Concentration-0.3M amine 50% N 0.3M Totalconcentration Methyl 0.3M pyrrollidone Polyvinyl alcohol n hydroxy 5norbornene 90% water 2.5 wt % Polyvinyl 2,3 di carboximide 10% Npyrrolidone 2.5 wt % Concentration-0.2M Methyl pyrrollidone Polyethylene glycol 80% water 5 wt % Poly 2 vinyl 20% N pyridine N oxideMethyl 5 wt % pyrrollidone Poly ethylene glycol 10% dimethyl monomethylether formamide 10 wt % Polyethylene glycol 80% water monomethylether20% N 5 wt % Poly vinyl Methyl alcohol 2.5 wt % pyrrollidone Poly vinylpyrrolidone 2.5 wt % Polyethylene glycol dimethyl ether 5 wt % Polyvinyl alcohol 2.5 wt % Poly vinyl pyrrolidone 2.5 wt % 3 Polyvinylalcohol- Boc- Asn-OH, Boc- 1-ethyl-3-(3- 50 wt % N methyl 100% Water 10wt % Asn(trt)-OH, Boc- dimethylaminopropyl) Pyrrolidone + 50 wt %Polyvinyl Asn(Xan)-OH carbodiimide) Di isopropyl ethyl 50% waterpyrrolidone-10 wt % Concentration- 0.2M and Concentration-0.3M amine 50%N 0.3M Total concentration Methyl 0.3M pyrrollidone Polyvinyl alcohol nhydroxy 5 norbornene 90% water 2.5 wt % Polyvinyl 2,3 di carboximide 10%N pyrrolidone 2.5 wt % Concentration-0.2M Methyl pyrrollidone Polyethylene glycol 80% water 5 wt % Poly 2 vinyl 20% N pyridine N oxideMethyl 5 wt % pyrrollidone Poly ethylene glycol 10% dimethyl monomethylether formamide 10 wt % Polyethylene glycol 80% water monomethylether20% N 5 wt % Poly vinyl Methyl alcohol 2.5 wt % pyrrollidone Poly vinylpyrrolidone 2.5 wt % Polyethylene glycol dimethylether 5 wt % Poly vinylalcohol 2.5 wt % Poly vinyl pyrrolidone 2.5 wt % 4 Polyvinyl alcohol-Boc- Asp(Obzl)-OH, 1-ethyl-3-(3- 50 wt % N methyl 100% Water 10 wt %Boc-Asp(Otbu)-OH, dimethylaminopropyl) Pyrrolidone + 50 wt % PolyvinylBoc- Asp-Otbu carbodiimide) Di isopropyl ethyl 50% water pyrrolidone-10wt % Concentration- 0.2M and Concentration-0.3M amine 50% N 0.3M Totalconcentration Methyl 0.3M pyrrollidone Polyvinyl alcohol n hydroxy 5norbornene 90% water 2.5 wt % Polyvinyl 2,3 di carboximide 10% Npyrrolidone 2.5 wt % Concentration-0.2M Methyl pyrrollidone Polyethylene glycol 80% water 5 wt % Poly 2 vinyl 20% N pyridine N oxideMethyl 5 wt % pyrrollidone Poly ethylene glycol 10% dimethyl monomethylether formamide 10 wt % Polyethylene glycol 80% water monomethylether20% N 5 wt % Poly vinyl Methyl alcohol 2.5 wt % pyrrollidone Poly vinylpyrrolidone 2.5 wt % Polyethylene glycol dimethylether 5 wt % Poly vinylalcohol 2.5 wt % Poly vinyl pyrrolidone 2.5 wt % 5 Polyvinyl alcohol-Boc- Cys(Acm)-OH, 1-ethyl-3-(3- 50 wt % N methyl 100% Water 10 wt %Boc-Cys(Bzl)-OH, Boc- dimethylaminopropyl) Pyrrolidone + 50 wt %Polyvinyl Cys(Acm)-Onp carbodiimide) Di isopropyl ethyl 50% waterpyrrolidone-10 wt % Concentration- 0.2M and Concentration-0.3M amine 50%N 0.3M Total concentration Methyl 0.3M pyrrollidone Polyvinyl alcohol nhydroxy 5 norbornene 90% water 2.5 wt % Polyvinyl 2,3 di carboximide 20%N pyrrolidone 2.5 wt % Concentration-0.2M Methyl pyrrollidone Polyethylene glycol 80% water 5 wt % Poly 2 vinyl 20% N pyridine N oxideMethyl 5 wt % pyrrollidone Poly ethylene glycol 10% dimethyl monomethylether formamide 10 wt % Polyethylene glycol 80% water monomethylether20% N 5 wt % Poly vinyl Methyl alcohol 2.5 wt % pyrrollidone Poly vinylpyrrolidone 2.5 wt % Polyethylene glycol dimethylether 5 wt % Poly vinylalcohol 2.5 wt % Poly vinyl pyrrolidone 2.5 wt % 6 Polyvinyl alcohol-Boc- Gln-OH, Boc- 1-ethyl-3-(3- 50 wt % N methyl 100% Water 10 wt %Gln(Trt)-OH, Boc- dimethylaminopropyl) Pyrrolidone + 50 wt % PolyvinylGln(Xan)-OH, Boc-Gln- carbodiimide) Di isopropyl ethyl 50% waterpyrrolidone-10 wt % Onp Concentration-0.3M amine 50% N Concentration-0.2M and Total concentration Methyl 0.3M 0.3M pyrrollidone Polyvinylalcohol n hydroxy 5 norbornene 90% water 2.5 wt % Polyvinyl 2,3 dicarboximide 10% N pyrrolidone 2.5 wt % Concentration-0.2M Methylpyrrollidone Poly ethylene glycol 80% water 5 wt % Poly 2 vinyl 20% Npyridine N oxide Methyl 5 wt % pyrrollidone Poly ethylene glycol 10%dimethyl monomethyl ether formamide 10 wt % Polyethylene glycol 80%water monomethylether 20% N 5 wt % Poly vinyl Methyl alcohol 2.5 wt %pyrrollidone Poly vinyl pyrrolidone 2.5 wt % Polyethylene glycoldimethylether 5 wt % Poly vinyl alcohol 2.5 wt % Poly vinyl pyrrolidone2.5 wt % 7 Polyvinyl alcohol- Boc- Glu-OH, Boc-Glu- 1-ethyl-3-(3- 50 wt% N methyl 100% Water 10 wt % NH2, Boc- Glu-Otbu, dimethylaminopropyl)Pyrrolidone + 50 wt % Polyvinyl Boc-Glu(Otbu)-OH carbodiimide) Diisopropyl ethyl 50% water pyrrolidone-10 wt % Concentration- 0.2M andConcentration-0.3M amine 50% N 0.3M Total concentration Methyl 0.3Mpyrrollidone Polyvinyl alcohol n hydroxy 5 norbornene 90% water 2.5 wt %Polyvinyl 2,3 di carboximide 10% N pyrrolidone 2.5 wt %Concentration-0.2M Methyl pyrrollidone Poly ethylene glycol 80% water 5wt % Poly 2 vinyl 20% N pyridine N oxide Methyl 5 wt % pyrrollidone Polyethylene glycol 10% dimethyl monomethyl ether formamide 10 wt %Polyethylene glycol 80% water monomethylether 20% N 5 wt % Poly vinylMethyl alcohol 2.5 wt % pyrrollidone Poly vinyl pyrrolidone 2.5 wt %Polyethylene glycol dimethylether 5 wt % Poly vinyl alcohol 2.5 wt %Poly vinyl pyrrolidone 2.5 wt % 8 Polyvinyl alcohol- Boc- Gly-OH,Boc-Gly- 1-ethyl-3-(3- 50 wt % N methyl 100% Water 10 wt % N(Ome)Me,Boc- Gly- dimethylaminopropyl) Pyrrolidone + 50 wt % Polyvinyl Osucarbodiimide) Di isopropyl ethyl 50% water pyrrolidone-10 wt %Concentration- 0.2M and Concentration-0.3M amine 50% N 0.3M Totalconcentration Methyl 0.3M pyrrollidone Polyvinyl alcohol n hydroxy 5norbornene 90% water 2.5 wt % Polyvinyl 2,3 di carboximide 10% Npyrrolidone 2.5 wt % Concentration-0.2M Methyl pyrrollidone Polyethylene glycol 80% water 5 wt % Poly 2 vinyl 20% N pyridine N oxideMethyl 5 wt % pyrrollidone Poly ethylene glycol 10% dimethyl monomethylether formamide 10 wt % Polyethylene glycol 80% water monomethylether20% N 5 wt % Poly vinyl Methyl alcohol 2.5 wt % pyrrollidone Poly vinylpyrrolidone 2.5 wt % Polyethylene glycol dimethylether 5 wt % Poly vinylalcohol 2.5 wt % Poly vinyl pyrrolidone 2.5 wt % 9 Polyvinyl alcohol-Boc- His-OH, Boc-His- 1-ethyl-3-(3- 50 wt % N methyl 100% Water 10 wt %Bom-OH, Boc- dimethylaminopropyl) Pyrrolidone + 50 wt % PolyvinylHis(Tos)-OH carbodiimide) Di isopropyl ethyl 50% water pyrrolidone-10 wt% Concentration- 0.2M and Concentration-0.3M amine 50% N 0.3M Totalconcentration Methyl 0.3M pyrrollidone Polyvinyl alcohol n hydroxy 5norbornene 90% water 2.5 wt % Polyvinyl 2,3 di carboximide 10% Npyrrolidone 2.5 wt % Concentration-0.2M Methyl pyrrollidone Polyethylene glycol 80% water 5 wt % Poly 2 vinyl 20% N pyridine N oxideMethyl 5 wt % pyrrollidone Poly ethylene glycol 10% dimethyl monomethylether formamide 10 wt % Polyethylene glycol 80% water monomethylether20% N 5 wt % Poly vinyl Methyl alcohol 2.5 wt % pyrrollidone Poly vinylpyrrolidone 2.5 wt % Polyethylene glycol dimethylether 5 wt % Poly vinylalcohol 2.5 wt % Poly vinyl pyrrolidone 2.5 wt % 10 Polyvinyl alcohol-Boc- Ile-OH 1-ethyl-3-(3- 50 wt % N methyl 100% Water 10 wt %Concentration- 0.2M and dimethylaminopropyl) Pyrrolidone + 50 wt %Polyvinyl 0.3M carbodiimide) Di isopropyl ethyl 50% water pyrrolidone-10wt % Concentration-0.3M amine 50% N Total concentration Methyl 0.3Mpyrrollidone Polyvinyl alcohol n hydroxy 5 norbornene 90% water 2.5 wt %Polyvinyl 2,3 di carboximide 10% N pyrrolidone 2.5 wt %Concentration-0.2M Methyl pyrrollidone Poly ethylene glycol 80% water 5wt % Poly 2 vinyl 20% N pyridine N oxide Methyl 5 wt % pyrrollidone Polyethylene glycol 10% dimethyl monomethyl ether formamide 10 wt %Polyethylene glycol 80% water monomethylether 20% N 5 wt % Poly vinylMethyl alcohol 2.5 wt % pyrrollidone Poly vinyl pyrrolidone 2.5 wt %Polyethylene glycol dimethylether 5 wt % Poly vinyl alcohol 2.5 wt %Poly vinyl pyrrolidone 2.5 wt % 11 Polyvinyl alcohol- Boc- Leu-OH1-ethyl-3-(3- 50 wt % N methyl 100% Water 10 wt % Concentration- 0.2Mand dimethylaminopropyl) Pyrrolidone + 50 wt % Polyvinyl 0.3Mcarbodiimide) Di isopropyl ethyl 50% water pyrrolidone-10 wt %Concentration-0.3M amine 50% N Total concentration Methyl 0.3Mpyrrollidone Polyvinyl alcohol n hydroxy 5 norbornene 90% water 2.5 wt %Polyvinyl 2,3 di carboximide 10% N pyrrolidone 2.5 wt %Concentration-0.2M Methyl pyrrollidone Poly ethylene glycol 80% water 5wt % Poly 2 vinyl 20% N pyridine N oxide Methyl 5 wt % pyrrollidone Polyethylene glycol 10% dimethyl monomethyl ether formamide 10 wt %Polyethylene glycol 80% water monomethylether 20% N 5 wt % Poly vinylMethyl alcohol 2.5 wt % pyrrollidone Poly vinyl pyrrolidone 2.5 wt %Polyethylene glycol dimethylether 5 wt % Poly vinyl alcohol 2.5 wt %Poly vinyl pyrrolidone 2.5 wt % 12 Polyvinyl alcohol- Boc- Lys-OH, Boc-1-ethyl-3-(3- 50 wt % N methyl 100% Water 10 wt % Lys(Z)-OH, Boc- Lys-dimethylaminopropyl) Pyrrolidone + 50 wt % Polyvinyl Osu carbodiimide)Di isopropyl ethyl 50% water pyrrolidone-10 wt % Concentration- 0.2M andConcentration-0.3M amine 50% N 0.3M Total concentration Methyl 0.3Mpyrrollidone Polyvinyl alcohol n hydroxy 5 norbornene 90% water 2.5 wt %Polyvinyl 2,3 di carboximide 10% N pyrrolidone 2.5 wt %Concentration-0.2M Methyl pyrrollidone Poly ethylene glycol 80% water 5wt % Poly 2 vinyl 20% N pyridine N oxide Methyl 5 wt % pyrrollidone Polyethylene glycol 10% dimethyl monomethyl ether formamide 10 wt %Polyethylene glycol 80% water monomethylether 20% N 5 wt % Poly vinylMethyl alcohol 2.5 wt % pyrrollidone Poly vinyl pyrrolidone 2.5 wt %Polyethylene glycol dimethylether 5 wt % Poly vinyl alcohol 2.5 wt %Poly vinyl pyrrolidone 2.5 wt % 13 Polyvinyl alcohol- Boc- Met-OH, Boc-1-ethyl-3-(3- 50 wt % N methyl 100% Water 10 wt % Met(O)-OHdimethylaminopropyl) Pyrrolidone + 50 wt % Polyvinyl Concentration- 0.2Mand carbodiimide) Di isopropyl ethyl 50% water pyrrolidone-10 wt % 0.3MConcentration-0.3M amine 50% N Total concentration Methyl 0.3Mpyrrollidone Polyvinyl alcohol n hydroxy 5 norbornene 90% water 2.5 wt %Polyvinyl 2,3 di carboximide 10% N pyrrolidone 2.5 wt %Concentration-0.2M Methyl pyrrollidone Poly ethylene glycol 80% water 5wt % Poly 2 vinyl 20% N pyridine N oxide Methyl 5 wt % pyrrollidone Polyethylene glycol 10% dimethyl monomethyl ether formamide 10 wt %Polyethylene glycol 80% water monomethylether 20% N 5 wt % Poly vinylMethyl alcohol 2.5 wt % pyrrollidone Poly vinyl pyrrolidone 2.5 wt %Polyethylene glycol dimethylether 5 wt % Poly vinyl alcohol 2.5 wt %Poly vinyl pyrrolidone 2.5 wt % 14 Polyvinyl alcohol- Boc- Phe-OH1-ethyl-3-(3- 50 wt % N methyl 100% Water 10 wt % Concentration- 0.2Mand dimethylaminopropyl) Pyrrolidone + 50 wt % Polyvinyl 0.3Mcarbodiimide) Di isopropyl ethyl 50% water pyrrolidone-10 wt %Concentration-0.3M amine 50% N Total concentration Methyl 0.3Mpyrrollidone Polyvinyl alcohol n hydroxy 5 norbornene 90% water 2.5 wt %Polyvinyl 2,3 di carboximide 10% N pyrrolidone 2.5 wt %Concentration-0.2M Methyl pyrrollidone Poly ethylene glycol 80% water 5wt % Poly 2 vinyl 20% N pyridine N oxide Methyl 5 wt % pyrrollidone Polyethylene glycol 10% dimethyl monomethyl ether formamide 10 wt %Polyethylene glycol 80% water monomethylether 20% N 5 wt % Poly vinylMethyl alcohol 2.5 wt % pyrrollidone Poly vinyl pyrrolidone 2.5 wt %Polyethylene glycol dimethylether 5 wt % Poly vinyl alcohol 2.5 wt %Poly vinyl pyrrolidone 2.5 wt % 15 Polyvinyl alcohol- Boc- Pro-OH,Boc-Pro- 1-ethyl-3-(3- 50 wt % N methyl 100% Water 10 wt % Omedimethylaminopropyl) Pyrrolidone + 50 wt % Polyvinyl Concentration- 0.2Mand carbodiimide) Di isopropyl ethyl 50% water pyrrolidone-10 wt % 0.3MConcentration-0.3M amine 50% N Total concentration Methyl 0.3Mpyrrollidone Polyvinyl alcohol n hydroxy 5 norbornene 90% water 2.5 wt %Polyvinyl 2,3 di carboximide 10% N pyrrolidone 2.5 wt %Concentration-0.2M Methyl pyrrollidone Poly ethylene glycol 80% water 5wt % Poly 2 vinyl 20% N pyridine N oxide Methyl 5 wt % pyrrollidone Polyethylene glycol 10% dimethyl monomethyl ether formamide 10 wt %Polyethylene glycol 80% water monomethylether 20% N 5 wt % Poly vinylMethyl alcohol 2.5 wt % pyrrollidone Poly vinyl pyrrolidone 2.5 wt %Polyethylene glycol dimethylether 5 wt % Poly vinyl alcohol 2.5 wt %Poly vinyl pyrrolidone 2.5 wt % 16 Polyvinyl alcohol- Boc- Ser-OH,Boc-Ser- 1-ethyl-3-(3- 50 wt % N methyl 100% Water 10 wt % OMe, Boc-Ser-Obzl, dimethylaminopropyl) Pyrrolidone + 50 wt % PolyvinylBoc-Ser(tbu)-OH carbodiimide) Di isopropyl ethyl 50% waterpyrrolidone-10 wt % Concentration- 0.2M and Concentration-0.3M amine 50%N 0.3M Total concentration Methyl 0.3M pyrrollidone Polyvinyl alcohol nhydroxy 5 norbornene 90% water 2.5 wt % Polyvinyl 2,3 di carboximide 10%N pyrrolidone 2.5 wt % Concentration-0.2M Methyl pyrrollidone Polyethylene glycol 80% water 5 wt % Poly 2 vinyl 20% N pyridine N oxideMethyl 5 wt % pyrrollidone Poly ethylene glycol 10% dimethyl monomethylether formamide 10 wt % Polyethylene glycol 80% water monomethylether20% N 5 wt % Poly vinyl Methyl alcohol 2.5 wt % pyrrollidone Poly vinylpyrrolidone 2.5 wt % Polyethylene glycol dimethylether 5 wt % Poly vinylalcohol 2.5 wt % Poly vinyl pyrrolidone 2.5 wt % 17 Polyvinyl alcohol-Boc- Thr-OH, Boc-Thr- 1-ethyl-3-(3- 50 wt % N methyl 100% Water 10 wt %OMe, Boc- Thr-Osu dimethylaminopropyl) Pyrrolidone + 50 wt % PolyvinylConcentration- 0.2M and carbodiimide) Di isopropyl ethyl 50% waterpyrrolidone-10 wt % 0.3M Concentration-0.3M amine 50% N Totalconcentration Methyl 0.3M pyrrollidone Polyvinyl alcohol n hydroxy 5norbornene 90% water 2.5 wt % Polyvinyl 2,3 di carboximide 10% Npyrrolidone 2.5 wt % Concentration-0.2M Methyl pyrrollidone Polyethylene glycol 80% water 5 wt % Poly 2 vinyl 20% N pyridine N oxideMethyl 5 wt % pyrrollidone Poly ethylene glycol 10% dimethyl monomethylether formamide 10 wt % Polyethylene glycol 80% water monomethylether20% N 5 wt % Poly vinyl Methyl alcohol 2.5 wt % pyrrollidone Poly vinylpyrrolidone 2.5 wt % Polyethylene glycol dimethylether 5 wt % Poly vinylalcohol 2.5 wt % Poly vinyl pyrrolidone 2.5 wt % 18 Polyvinyl alcohol-Boc- Trp-OH, Boc- 1-ethyl-3-(3- 50 wt % N methyl 100% Water 10 wt %Trp(For)-OH dimethylaminopropyl) Pyrrolidone + 50 wt % PolyvinylConcentration- 0.2M and carbodiimide) Di isopropyl ethyl 50% waterpyrrolidone-10 wt % 0.3M Concentration-0.3M amine 50% N Totalconcentration Methyl 0.3M pyrrollidone Polyvinyl alcohol n hydroxy 5norbornene 90% water 2.5 wt % Polyvinyl 2,3 di carboximide 10% Npyrrolidone 2.5 wt % Concentration-0.2M Methyl pyrrollidone Polyethylene glycol 80% water 5 wt % Poly 2 vinyl 20% N pyridine N oxideMethyl 5 wt % pyrrollidone Poly ethylene glycol 10% dimethyl monomethylether formamide 10 wt % Polyethylene glycol 80% water monomethylether20% N 5 wt % Poly vinyl Methyl alcohol 2.5 wt % pyrrollidone Poly vinylpyrrolidone 2.5 wt % Polyethylene glycol dimethylether 5 wt % Poly vinylalcohol 2.5 wt % Poly vinyl pyrrolidone 2.5 wt % 19 Polyvinyl alcohol-Boc- Tyr-OH, Boc- 1-ethyl-3-(3- 50 wt % N methyl 100% Water 10 wt %Tyr(Me)-OH, Boc- Tyr- dimethylaminopropyl) Pyrrolidone + 50 wt %Polyvinyl Otbu, Boc-Tyr(Bzl)-OH carbodiimide) Di isopropyl ethyl 50%water pyrrolidone-10 wt % Concentration- 0.2M and Concentration-0.3Mamine 50% N 0.3M Total concentration Methyl 0.3M pyrrollidone Polyvinylalcohol n hydroxy 5 norbornene 90% water 2.5 wt % Polyvinyl 2,3 dicarboximide 10% N pyrrolidone 2.5 wt % Concentration-0.2M Methylpyrrollidone Poly ethylene glycol 80% water 5 wt % Poly 2 vinyl 20% Npyridine N oxide Methyl 5 wt % pyrrollidone Poly ethylene glycol 10%dimethyl monomethyl ether formamide 10 wt % Polyethylene glycol 80%water monomethylether 20% N 5 wt % Poly vinyl Methyl alcohol 2.5 wt %pyrrollidone Poly vinyl pyrrolidone 2.5 wt % Polyethylene glycoldimethylether 5 wt % Poly vinyl alcohol 2.5 wt % Poly vinyl pyrrolidone2.5 wt % 20 Polyvinyl alcohol- Boc- Val-OH 1-ethyl-3-(3- 50 wt % Nmethyl 100% Water 10 wt % Concentration- 0.2M and dimethylaminopropyl)Pyrrolidone + 50 wt % Polyvinyl 0.3M carbodiimide) Di isopropyl ethyl50% water pyrrolidone-10 wt % Concentration-0.3M amine 50% N Totalconcentration Methyl 0.3M pyrrollidone Polyvinyl alcohol n hydroxy 5norbornene 90% water 2.5 wt % Polyvinyl 2,3 di carboximide 10% Npyrrolidone 2.5 wt % Concentration-0.2M Methyl pyrrollidone Polyethylene glycol 80% water 5 wt % Poly 2 vinyl 20% N pyridine N oxideMethyl 5 wt % pyrrollidone Poly ethylene glycol 10% dimethyl monomethylether formamide 10 wt % Polyethylene glycol 80% water monomethylether20% N 5 wt % Poly vinyl Methyl alcohol 2.5 wt % pyrrollidone Poly vinylpyrrolidone 2.5 wt % Polyethylene glycol dimethylether 5 wt % Poly vinylalcohol 2.5 wt % Poly vinyl pyrrolidone 2.5 wt % Note: The % in thesolvent column refers to the contents of the solvent itself. E.g., 100%water means that the solvent is 100% water.

Example 4 Yield Data for 20-Mer Homopolymeric Peptides

The materials and methods used in this example are as described above inExamples 1-3. Specifically, amino acids Ala, Asp and His were preparedas follows. The inert water soluble polymers (3% by weight PVP and 7% byweight PVA) were dissolved in the ratio of 1:2.5 in deionized (DI) waterwhich makes up about 10% by weight of the solution along with 2% byweight amino acid concentration. 4% by weight for each EDC and HonB wereadded as reagents twice the concentration of amino acids. DIEA (4% byweight) was added at the same concentration as EDC and HoNb. This watercoupling solution was spin coated on a derivatized substrate to form auniform solid layer, all available to couple to the substrate. The waferwas then baked on a hot plate for 2 minutes at 90° C. to remove theremaining solvent (DI water) and coupled at the same time. Next thecoupling coat was washed away with DI water in a strip module.

Capping Solution was Prepared as Follows:

Acetic anhydride was obtained from Sigma Aldrich Corp. PVP was dissolvedin N methyl pyrrolidone which makes up 2% by weight (1-2% by weight wastested and gave similar results; data not shown) of the total solution.Next acetic anhydride was added to make up 25% by weight (20-30% byweight was tested and gave similar results; data not shown) of thesolution. This capping solution was then spin coated on the wafers in acapping module by spinning the wafers at 2000 rpm for 30 sec. The waferswere then baked in a cap bake module for up to 2 minutes at 75° C. tocomplete the capping process. The remaining solution was washed awaywith DI water in a strip module.

The same steps were followed for synthesizing 20-mer peptides with thefollowing sequences:

1.  (SEQ ID NO: 2) Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala, 2.  (SEQ ID NO: 3)Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp, 3.  (SEQ ID NO: 4)His-His-His-His-His-His-His-His-His-His-His-His-His-His-His-His-His-His-His-His.

This example shows the step yield data for each of the above 20-meramino acid sequences. To measure step yield via fluorescence, afluorescent dye molecule was coupled to the sequence of amino acids inorder to determine the coupling efficiency. The amount of fluoresceindye coupled gives a direct measure of the amount of sequence grown.

The formula used to calculate step yield was: Stepyield=(F_(n)/F₁)^(1/n−1), where F₁ and F_(n) denotes the fluoresceincoupling intensity read out from a fluorescent scanner device at thefirst step and the nth step. The coupling yield was calculated using theformula E=10 ̂ ((log F)/C) where F equals fraction of full length andC=number of couplings=length −1.

Table 3 shows the fluorescence signal intensity at each layer. FIG. 4Ashows the readout of the fluorescence signal from each of the 20-merexperiments. FIG. 4B shows a graph of fluorescence signal intensity vs.each amino acid layer. FIG. 5 shows a graph of overall step yield vs.each amino acid layer. Table 4 shows the yield efficiency for eachcoupling step. The coupling efficiency of each amino acid was calculatedto be greater than 98% in each instance across the entire length of eachof the 20-mer peptides.

TABLE 3 Fluorescence signal intensity at each layer for PolyA, PolyD,and PolyH Amino Acid 1-mer 2-mer 3-mer 4-mer 5-mer 6-mer 7-mer 8mer9-mer 10-mer A 41000   40363.4 39679   38924   38261   37496.8 36745.936012.2 35291.1 34583.6 D 48000   47298.1 46577.6 45823.1 45123.544445.8 43787.9 43132.2 42484.7 41846.3 H 25006.5 24623.4 24253.423899.7 23541.2 23187.6 22839.9 22496.4 22158.7 21825.4 11-mer 12-mer13-mer 14-mer 15-mer 16-mer 17-mer 18-mer 19-mer 20-mer A 33891.233212.3 32547.5 31995.6 31556.7 30930.9 30517.9 29823.6 28965.4 28554.4D 41213.4 40594.5 39986.7 39386.3 38798.2 38215.4 37642.1 37067.636542.3 35995.6 H 21497.8 21175.4 20857.6 20544.3 20234.5 19932.219632.2 19338.1 19047.9 18762.3

TABLE 4 Yield efficiency at each layer for PolyA, PolyD, and PolyH 1-mer2-mer 3-mer 4-mer 5-mer 6-mer 7-mer 8-mer 9-mer 10-mer Couplings 1 2 3 45 6 7 8 9 A amino 1 0.984 0.968 0.949 0.933 0.915 0.896 0.878 0.8610.844 acid Coupling 0.984 0.984 0.983 0.983 0.982 0.982 0.982 0.9810.981 Efficiency D amino 1 0.985 0.970 0.955 0.940 0.926 0.912 0.8990.885 0.872 acid Coupling 0.985 0.985 0.985 0.985 0.985 0.985 0.9850.985 0.985 Efficiency H amino 1 0.985 0.970 0.956 0.941 0.927 0.9130.900 0.886 0.873 acid Coupling 0.985 0.985 0.9855 0.9855 0.9855 0.98550.9855 0.985 0.985 Efficiency 11-mer 12-mer 13-mer 14-mer 15-mer 16-mer17-mer 18-mer 19-mer 20-mer Couplings 10 11 12 13 14 15 16 17 18 19 Aamino 0.827 0.81 0.794 0.780 0.770 0.754 0.744 0.727 0.706 0.696 acidCoupling 0.981 0.981 0.981 0.981 0.981 0.981 0.982 0.981 0.981 0.981Efficiency D amino 0.859 0.846 0.833 0.821 0.808 0.796 0.784 0.772 0.7610.750 acid Coupling 0.985 0.985 0.985 0.985 0.985 0.985 0.985 0.9850.985 0.985 Efficiency H amino 0.860 0.847 0.834 0.822 0.809 0.797 0.7850.773 0.762 0.750 acid Coupling 0.985 0.985 0.985 0.985 0.985 0.9850.985 0.985 0.985 0.985 Efficiency

Example 5 Yield Data for Synthesis of 1 to 12-Mer Peptides

Materials and Methods:

Water soluble inert polymers (such as poly vinyl alcohol (PVA),polyvinyl pyrrolidone (PVP), or poly ethylene glycol) were obtained fromPolysciences Inc. EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide),and HonB (n-hydroxy-5-norbornene-2,3-di carboximide) were obtained fromSigma Aldrich Corp. DIEA (Diisopropylethylamine) was obtained from SigmaAldrich Corp. All t-boc/Fmoc protected amino acids were obtained fromAAPPTEC/Anaspec.

The inert water soluble polymers (3% by weight PVP and 7% by weight PVA)were dissolved in the ratio of 1:2.5 in deionized (DI) water which makesup about 10% by weight of the solution along with 2% by weight aminoacid concentration. Amino acids used in this example were Boc-Lys-OH,Boc-Leu-OH, Boc-Met-OH, Boc-Thr-OH, Boc-Ser-OH, Boc-Asp-OH, Boc-Gly-OH,Boc-Ile-OH, Boc-Ala-OH, Boc-Arg-OH, Boc-Val-OH.

4% by weight of each EDC and HonB were added as reagents twice theconcentration of amino acids. 4% by weight DIEA was added at the sameconcentration as EDC and HoNb.

Capping Solution was Prepared as Follows:

Acetic anhydride was obtained from Sigma Aldrich Corp. PVP was dissolvedin N-methylpyrrolidone which makes up 2% by weight (1-2% by weight werealso tested, data not shown) of the total solution. Next aceticanhydride was added to make up 25% by weight (20-30% by weight were alsotested, data not shown) of the solution.

Fluorescein Coupling Solution is Prepared as Follows:

5,6 FAM Carboxyfluorescein was obtained from Anaspec. 0.1M Boc-Gly-OH(from AAPPTeC), 0.05M 5,6 FAM and 0.1M HoNb (Sigma Aldrich) and 0.1M EDC(Sigma Aldrich) was dissolved in water along with 5% by weightPolyvinylpyrrollidone (PolySciences).

The derivatized wafer (see Example 1) was coated with the above resistat 2000 rpm and baked at 85° C. for 90 secs. The wafer was exposed in aNikon S-203 scanner and then post baked at 75° C. for 120 secs. Theresist was stripped with deionized water. The first coupling solutionwith amino acid Boc-Lys-OH was spin coated on a derivatized substrate toform a solid layer, all available to couple to the substrate. The waferwas then baked on a hot plate for 2 minutes at 90° C. to remove theremaining solvent (DI water) and coupled at the same time. Next thecoupling coat was washed away with DI water in a strip module.

This capping solution was then spin coated on the wafers in a cappingmodule by spinning the wafers at 2000 rpm for 30 seconds. The waferswere then baked in a cap bake module for up to 2 minutes at 75° C. tocomplete the capping process. The remaining solution was washed awaywith DI water in a strip module.

The same procedure was followed for the other amino acids in thesequence Lys-Leu-Glu-Arg-Ser-Thr-Val-Met-Ile-Lys-Gly-Asp (SEQ ID NO: 5).

All peptide lengths between 1 and 12 were synthesized for the abovepeptide. After the desired peptide sequence was synthesized, thefluorescein coupling solution was spin coated on the wafer at 2,000 rpmto form a coupling dye coat. Then the wafers were baked at 65° C. for 2mins and then the dye solution was washed away with water. Thiscompleted the coupling of fluorescein dye to allow measurement of thesignals. The signal was then read off a fluorescence microscope.

Results:

The image in FIG. 6 shows the readout of the fluorescence signal fromthe 1 to 12-mer experiment and has 4 rows and 12 columns. Each of the 4rows represent the same sequence. The columns contain the sequencesynthesized such that one amino acid is added in each column. From leftto right, each column thus represents the following sequences:

1. Lys, 2. Lys-Leu, 3.  Lys-Leu-Glu, 4. (SEQ ID NO: 6) Lys-Leu-Glu-Arg,5.  (SEQ ID NO: 7) Lys-Leu-Glu-Arg-Ser, 6.  (SEQ ID NO: 8)Lys-Leu-Glu-Arg-Ser-Thr, 7.  (SEQ ID NO: 9) Lys-Leu-Glu-Arg-Ser-Thr-Val,8.  (SEQ ID NO: 10) Lys-Leu-Glu-Arg-Ser-Thr-Val-Met, 9.  (SEQ ID NO: 11)Lys-Leu-Glu-Arg-Ser-Thr-Val-Met-Ile, 10.  (SEQ ID NO: 12)Lys-Leu-Glu-Arg-Ser-Thr-Val-Met-Ile-Lys, 11.  (SEQ ID NO: 13)Lys-Leu-Glu-Arg-Ser-Thr-Val-Met-Ile-Lys-Gly, 12.  (SEQ ID NO: 5)Lys-Leu-Glu-Arg-Ser-Thr-Val-Met-Ile-Lys-Gly-Asp.

The coupling yield was calculated using the formula E=10 ̂ ((log F)/C)where F equals fraction of full length and C=number of couplings=length−1. The formula used to calculate step yield was: Stepyield=(F_(n)/F₁)^(1n−1), where F₁ and F_(n) denotes the fluoresceincoupling intensity read out from a fluorescent scanner device at thefirst step and the nth step.

Table 5 shows the fluorescence signal intensity, the yield efficiencyfor each coupling step, and the overall yield.

TABLE 5 1 mer 2 mer 3 mer 4 mer 5 mer 6 mer Peptide K KL KLE KLER KLERSKLERST Sequence (SEQ ID  (SEQ ID (SEQ ID NO: 6) NO: 7) NO: 8)Fluorescence 54000 53298 52461.2 51522.1 50654 50032 signal intensityYield for 1 0.987 0.986 0.984 0.984 0.985 each coupling Overall 1 0.9870.972 0.954 0.938 0.927 yield 7 mer 8 mer 9 mer 10 mer 11 mer 12 merPeptide KLERST KLERSTV KLERSTVM KLERSTVMI KLERSTVMIK KLERSTVMIKGSequence V M I K G D (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ IDNO: 9) NO: 10) NO: 11) NO: 12) NO: 13) NO: 5) Fluorescence 49234 4833947456 46999 46097 45223.3 signal intensity Yield for 0.985 0.9845 0.9840.985 0.984 0.984 each coupling Overall 0.912 0.895 0.879 0.870 0.8540.837 yield

The coupling efficiency of each amino acid was calculated to be greaterthan 98% in each instance across the entire 12-mer peptide and theoverall yield of the 12 amino acid peptide was calculated as 83.74%. SeeTable 5.

The process above was repeated with a 12-mer homopolymeric peptideAAAAAAAAAAAA (SEQ ID NO: 14). The coupling formulation used for thispeptide synthesis is from Table 2 and comprised Boc-Ala-OH. Couplingyield was determined from fluorescence intensity at each synthesis step,as described above. Table 6 shows the fluorescence signal intensity, theyield efficiency for each coupling step, and the overall yield at eachstep of synthesis of the polyA peptide.

TABLE 6 1 mer 2 mer 3 mer 4 mer 5 mer 6 mer Peptide A AA AAA AAAA AAAAAAAAAAA Sequence (SEQ ID (SEQ ID (SEQ ID NO: 15) NO: 16) NO: 17)Fluorescence 61000 60363.4 59558.98 58826 58231 57436.8 signal intensityYield for 1 0.990 0.988 0.988 0.988 0.988 each coupling Overall 1 0.9900.976 0.964 0.955 0.942 yield  7 mer 8 mer 9 mer 10 mer 11 mer 12 merPeptide AAAAAAA AAAAAAAA AAAAAAAAA AAAAAAAAAA AAAAAAAAAAA AAAAAAAAAAAASequence (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID  (SEQ ID NO: 18)NO: 19) NO: 20) NO: 21) NO: 22) NO: 14) Fluorescence 56705.9 56001.2355289.1 54576.6 53888.2 53212.3 signal intensity Yield for 0.988 0.9880.988 0.988 0.988 0.988 each coupling Overall 0.930 0.918 0.906 0.8950.883 0.872 yield

Examples 4 and 5 illustrate a key advantage of the present invention;coupling efficiency >98% is stably maintained for each step of peptidesynthesis. Because the coupling efficiency is high and stable, eachfeature has a relatively greater fraction of intended full lengthpeptide and relatively lower fraction of less than full lengthcontaminants. For example, more than 80% of the peptide moleculescomprising a 12-mer peptide feature would be full length, and 70% ofmolecules in a 20-mer peptide feature would be full length. As wedemonstrate in Example 6, this is a remarkable advance of the prior artmethods.

Example 6 Comparison with Prior Art Array Synthesis

Materials and Methods:

We followed a standard prior art peptide array synthesis based on theMerrifield solid phase peptide synthesis [(R. B. Merrifield (1963).“Solid Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide”. J.Am. Chem. Soc. 85 (14): 2149-2154) Pellois, J P et al., “Individuallyaddressable parallel peptide synthesis on microchips”. Nat Biotechnology2002 September: 20(9):922-6). We determined the coupling efficiency foreach step. All Boc-protected amino acids and Hobt (hydroxybenzotriazole)were obtained from AAPPTEC. Wafers with silicon oxide were purchasedfrom University Wafers. Diisopropyl carbodiimide was obtained fromCreosalus. 99% by weight sulfuric acid in water and 33% by weighthydrogen peroxide in water are obtained from Sigma Aldrich. Piranhasolution was prepared by mixing a solution with a final concentration of50% sulfuric acid and 50% hydrogen peroxide. Aminopropyl triethoxysilaneand ethanol are obtained from Sigma Aldrich. Dimethyl formamide (DMF),Dichloromethane (DCM), acetic anhydride, N-methyl-2-pyrrolidinone (NMP),and acetone and isopropyl alcohol solution (IPA) were obtained from VWR.Polymethylmethacrylate (PMMA) was obtained from Polysciences Inc.,Bis(4-tert-butyl phenyl) Iodonium Hexafluoroantimonate (Photoacidgenerator (PAG) was obtained from Hampford Research.Isopropylthioxanthenone (ITX) was obtained from Sigma Aldrich. PGMEA(propylene glycol methyl ether acetate) was obtained from Alfa Aesar.Diisopropyl ethylamine (DIEA) was obtained from Alfa Aesar.

Wafers were cleaned with piranha solution. These wafers are dipped with0.5% aminopropyltriethoxy silane in ethanol for 15 minutes and thenwashed with ethanol. Now the wafers are cured at 110° C. for 30 minutesin a nitrogen bake oven. Amino acid and activator solution was preparedas follows: 0.1M Boc-Gly-OH, 0.1M DIC and 0.1M Hobt were dissolved inN-methyl-2-pyrrolidinone. The wafers were then immersed in this solutionfor 30 minutes in a vessel to form the first layer. The wafers were thenwashed with DCM/DMF (1:1,v/v), DMF, DCM, and DMF in sequencerespectively by shaking in a vessel for 5 minutes each at 100 rpm. 50%by weight acetic anhydride solution in DMF was then added to the wafersfor capping. The wafers were then washed with DMF and IPA.

Heteropolymeric Sequence:

We prepared a photoresist solution of 2.5% by weight PMMA, 5% by weightPAG and 5% by weight ITX dissolved in PGMEA (88.5% by weight). Thisresist formulation was spin coated at 2000 rpm for 60 seconds and thenpost baked at 85° C. for 90 seconds. The wafers were then cooled at 23°C. for 5 minutes. These wafers were then exposed at 40 mJ/cm² with onereticle over the whole wafer in deep UV scanner. Next, the wafers werebaked at 65° C. for 1 min. on a hot plate. The wafers were then soakedwith Acetone and DI water for 2 minutes each. They were then air driedwith nitrogen. The wafer was washed with 5% DIEA in DMF for 10 minutes.

All Boc-amino acids were obtained from AAPPTEC and was mixed with 0.1MDIC and 0.1M Hobt and dissolved in NMP. The wafers were then immersedwith this solution for 30 mins followed by wash with DCM/DMF as instep 1. Then the wafers were capped with 50% Acetic anhydride/DMFsolution for 30 mins. The non homopolymeric sequence was synthesized asKLERSTVMIKGD (SEQ ID NO: 5).

Coupling Yield Test:

5,6 FAM Carboxyfluorescein was obtained from Anaspec. 0.1M Boc-Gly-OH(from AAPPTeC), 0.1M Hobt (Sigma Aldrich) and 0.1M DIC (Creosalus) wasdissolved in NMP. The wafers were dipped in this solution covered indark for 1 hour to detect the coupling yield.

The coupling yield was calculated using the formula E=10 ̂ ((log F)/C)where F equals fraction of full length and C=number of couplings=length−1. The formula used to calculate step yield was: Stepyield=(F_(n)/F₁)^(1/n−1), where F₁ and F_(n) denotes the fluoresceincoupling intensity read out from a fluorescent scanner device at thefirst step and the nth step.

FIG. 7A shows an image of the chip with intensity from left to rightprovides a measure of efficiency of total synthesis from 1 to 12peptides of the ‘KLERSTVMIKGD’ (SEQ ID NO: 5) peptide in duplicate (2series) (bottom) and intensity profile graph for each spot (top). Table7 shows the numerical value of fluorescence signal intensity from FIG.7A, the yield efficiency for each coupling step, and the overall yield.FIG. 8A shows a plot of the stepwise synthesis overall yield of thepeptide ‘KLERSTVMIKGD’ (SEQ ID NO: 5) on a chip using the Vibrant chipand method described above (results in Table 5) vs. the standardsynthesis of a chip (results in Table 7). FIG. 8C shows plot of stepwisesynthesis coupling efficiency of the peptide ‘KLERSTVMIKGD’ (SEQ ID NO:5) on a chip using the Vibrant chip and method described above (resultsin Table 5) vs. the standard synthesis of a chip (results in Table 7).As can be seen from the results, the efficiency of synthesis issignificantly improved for peptides greater than 3 amino acids in lengthusing our method.

TABLE 7 1 mer 2 mer 3 mer 4 mer 5 mer 6 mer Peptide K KL KLE KLER KLERSKLERST Sequence (SEQ ID  (SEQ ID (SEQ ID NO: 6) NO: 7) NO: 8)Fluorescence 58000 57500 57000 48000 40500 26200 signal intensityYield for 1 0.991 0.991 0.939 0.914 0.853 each coupling Overall 1 0.9910.982 0.828 0.698 0.452 yield 7 mer 8 mer 9 mer 10 mer 11 mer 12 merPeptide KLERST KLERSTV KLERSTVM KLERSTVMI KLERSTVMIK KLERSTVMIKGSequence V M I K G D (SEQ ID (SEQ ID (SEQ ID  (SEQ ID  (SEQ ID (SEQ ID NO: 9) NO: 10) NO: 11) NO: 12) NO: 13) NO: 5) Fluorescence 19000 1400012500 12500 10000 10000 signal intensity Yield for 0.830 0.816 0.8250.843 0.839 0.852 each coupling Overall  0.328 0.241 0.216 0.216 0.1720.172 yield

Homopolymeric Sequence:

We prepared a photoresist solution of 2.5% by weight PMMA, 5% by weightPAG and 5% weight ITX dissolved in PGMEA (88.5% by weight). This resistwas spin coated at 2000 rpm for 60 seconds and then post baked at 85° C.for 90 seconds. The wafers were then cooled at 23° C. for 5 minutes.These wafers were then exposed at 40 mJ/cm² with one reticle over thewhole wafer in deep UV scanner. Next, the wafers were baked at 65° C.for 1 minute on a hot plate. The wafers were then soaked with acetoneand DI water for 2 minutes each. They were then air dried with nitrogen.The wafer was then washed with 5% DIEA in DMF for 10 minutes.

0.1M Boc-Ala-OH obtained from AAPPTEC was mixed with 0.1M DIC and 0.1MHobt and dissolved in NMP. The wafers were then immersed with thissolution for 30 minutes followed by wash with DCM/DMF as in step 1. Thenthe wafers were capped with 50% Acetic anhydride/DMF solution for 30minutes.

These steps were followed for all the 12 layers of Alanine.

Coupling Yield Test:

5,6 FAM Carboxyfluorescein was obtained from Anaspec. 0.1M Boc-Gly-OH(from AAPPTeC), 0.1M Hobt (Sigma Aldrich) and 0.1M DIC (Creosalus) wasdissolved in NMP. The wafers were dipped in this solution covered indark for 1 hour to detect the coupling yield.

The coupling yield was calculated using the formula E=10 ̂ ((log F)/C)where F equals fraction of full length and C=number of couplings=length−1. The formula used to calculate step yield was: Stepyield=(F_(n)/F₁)^(1n−1), where F₁ and F_(n) denotes the fluoresceincoupling intensity read out from a fluorescent scanner device at thefirst step and the nth step.

FIG. 7B shows an image of the chip with intensity from left to rightprovides a measure of efficiency of total synthesis from 1 to 12peptides of the polyA peptide in duplicate (2 series) and intensityprofile graph for each spot. Table 8 shows the numerical value offluorescence signal intensity from FIG. 7B, the yield efficiency foreach coupling step, and the overall yield. FIG. 8B shows a plot of thepolyA synthesis on a chip using the Vibrant chip and method describedabove (results in Table 6) vs. the standard synthesis of a chip (resultsin Table 8). FIG. 8D shows plot of stepwise synthesis couplingefficiency of the polyA synthesis on a chip using the Vibrant chip andmethod described above (results in Table 6) vs. the standard synthesisof a chip (results in Table 8). As can be seen from the results, theefficiency of synthesis is significantly improved for peptides greaterthan 3 amino acids in length using our method.

TABLE 8 1 mer 2 mer 3 mer 4 mer 5 mer 6 mer Peptide A AA AAA AAAA AAAAAAAAAAA Sequence (SEQ ID (SEQ ID (SEQ ID NO: 15) NO: 16) NO: 17)Fluorescence 62000 61500 61000 57500 35400 30000 signal intensityYield for 1 0.992 0.992 0.975 0.869 0.864 each coupling Overall 1 0.9920.984 0.927 0.571 0.484 yield 7 mer 8 mer 9 mer 10 mer 11 mer 12 merPeptide AAAAAAA AAAAAAAA AAAAAAAAA AAAAAAAAAA AAAAAAAAAAA AAAAAAAAAAAASequence (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 18) NO: 19)NO: 20) NO: 21) NO: 22) NO: 14) Fluorescence 20000 14000 12500 1250010000 10000 signal intensity Yield for 0.828 0.808 0.819 0.837 0.8330.847 each coupling Overall 0.323 0.226 0.202 0.202 0.161 0.161 yield

Example 7 Coupling of Amino Acids to a Substrate

This example provides the typical signature of how the coupling stepsperform on a microarray platform using all 20 amino acids in water-basedcoupling solutions on two substrates (i.e., wafers) and signal intensityas a read-out. This test was run as a quality control on each batch ofwafer (substrate) to match the performance. For each experiment, themeasured signal intensity gives a direct relation of the coupling yield.

Derivatized substrate was obtained as described in Example 1. Allcouplings of amino acids mentioned in this example were coupled asdescribed below. The same steps were repeated on all wafers done in abatch. This is a method of performing quality control after completingthe manufacturing of an entire batch process of wafers.

All 20 water based coupling solutions were prepared as follows: Theinert water soluble polymers (3% by weight PVP and 7% by weight PVA)were dissolved in the ratio of 1:2.5 in deionized (DI) water which makesup about 10% by weight of the solution along with 2% by weight aminoacid concentration. 4% by weight of each EDC and HonB were added asreagents twice the concentration of amino acids. 4% by weight DIEA wasadded at the same concentration as EDC and HoNb. This water couplingsolution was spin coated on a derivatized substrate to form a uniformsolid layer all available to couple to the substrate. The wafer was thenbaked on a hot plate for 2 minutes at 90° C. to remove the remainingsolvent (DI water) and coupled at the same time. Next the coupling coatwas washed away with DI water in a strip module.

5,6 fAM Carboxy fluorescein was obtained from Anaspec. 0.1M Boc-Gly-OH(from AAPPTeC), 0.05M 5,6FAm and 0.1M HoNb (Sigma Aldrich) and 0.1M EDC(Sigma Aldrich) was dissolved in water along with 5% by weight Polyvinyl pyrrollidone (PolySciences). This solution is called thefluorescein coupling solution. This solution was spin coated on thewafer at 2000 rpm to form a coupling dye coat. Then the wafers werebaked at 65° C. for 2 mins and then the dye solution was washed awaywith water. This completes the coupling of fluorescein dye to measurethe signals. The signal was then read off a fluorescence microscope.

Capping Solution was Prepared as Follows:

Acetic anhydride was obtained from Sigma Aldrich Corp. PVP was dissolvedin N methyl pyrrolidone which makes up about 1-2% of the total solution,thus the contents of the solution were as follows: PVP— 1-2% by weight.Acetic Anhydride 20-30% by weight and the remainder was N methylpyrrollidone. Then acetic anhydride was added to make up about 20-30% byweight of the solution. This capping solution was spin coated on thewafers in a capping module by spinning the wafers at 2000 rpm for 30sec. The wafers were then baked in a cap bake module for up to 2 minutesat 75° C. to complete the capping process. The remaining solution waswashed away with DI water in a strip module.

The above procedure was followed for coupling each amino acid. Thus togrow a 12-mer amino acid peptide chain using all the twenty amino acidsuses 240 steps. Fluorescein was coupled as explained above to read thedata out from the fluorescein scanner (Nikon AIR Confocal Scanner).

In this example, amino acidsAla-Cys-Asp-Glu-Phe-Gly-His-Ile-Lys-Leu-Met-Asn-Pro-Gln-Arg-Ser-Thr-Val-Trp-Tyr(SEQ ID NO: 60) were used to synthesize and fill the microarray usingthe procedures described above. FIG. 9 shows the fluorescence signalintensity of each amino acid at each layer. Amino acids His, Asn, andTrp showed a lower value due to fluorescein quenching. The graph in FIG.9 shows an upward curve after these amino acids hence showing highcoupling yield on every synthesis step.

FIG. 10 shows the normalized signal intensity for all twenty amino acidsgrown at each layer (total of 12 layers) on each of the wafers tested.Normalized data=(Raw fluorescence signal intensity/(sum value of eachlayer/Average of fluorescence signal intensity sum across all layers)).Thus, coupling yield (as measured via signal intensity) remains highacross each amino acid and each layer, with the exception of His, Asn,and Trp due to fluorescein quenching.

Example 8 Testing of Water-Based Photoactive Formulations

Materials and Methods

Polymers—polyethylene glycol monomethyl ether, Polyvinyl pyrrollidone,Poly(2-dimethylaminoethyl methacrylate), Poly(2-hydroxypropylmethacrylate), Poly 4 vinyl pyridine were obtained from Polysciences.

Photoacid generators 4 Methoxyphenyl)phenyliodoniumtrifluoromethanesulfonate, (4 methoxyphenyl)dimethylsulfonium triflate,(2,4-dihydroxyphenyl)dimethylsulfonium triflate were obtained fromHamilton Research Inc.

Isopropyl thioxanthenone and ethyl lactate was obtained from SigmaAldrich.

Photoactive Formulations:

Water resist-1 was prepared by mixing the polymer polyethylene glycolmonomethyl ether (2% by weight) and polyvinyl pyrrollidone (2% byweight) in water and letting it dissolve overnight. 4Methoxyphenyl)phenyliodonium trifluoromethanesulfonate (5% by weight)was added along with isopropyl thioxanthenone (5% by weight) anddissolved overnight. Water resist-2 was prepared by mixing the polymerPoly(2-dimethylaminoethyl methacrylate) (2.5% by weight) in a solvent ofwater (90% by weight) and ethyl lactate (10% by weight) and letting itdissolve overnight. (4 methoxyphenyl)dimethylsulfonium triflate (5% byweight) was added along with isopropyl thioxanthenone and dissolvedovernight.

For all other resists—water and non-water were prepared the same way asdescribed above. Letting the polymer dissolve overnight in thecorresponding solvent and then adding the photoacid generator and/orphotoinitiator in the specified amounts.

Substrates were obtained as described in Example 1. Then wafers weresurface derivatized. Once the resists were prepared, the resists werespin coated onto the substrate at speeds varying from 1000 rpm to 2000rpm to obtain the desired thickness. For example, water resist 1 wasspun at 2000 rpm to obtain a 0.2 μm thickness. The wafers were thenexposed in a Nikon S 203 scanner for varying exposure energy from 6mJ/cm² to 26 mJ/cm². Next the wafers were post baked at 85° C. in a hotplate for 2 mins and then the resist was stripped with water.

EDC —1-ethyl-3-(3-dimethylaminopropyl) carbodiimide), and HonB—n hydroxy5 norbornene 2,3 di carboximide were obtained from Sigma Aldrich corp.DIEA—Di isopropyl ethylamine was obtained from Sigma Aldrich Corp.

Boc-Gly-OH was obtained from AAPPTEC/Anaspec. The inert water solublepolymer PVP and PVA were dissolved in the ratio of 1:2.5 in deionized(DI) water which makes up about 10% by weight of the solution along with1-2% by weight amino acid concentration. EDC and HonB were added asreagents at 2× the percentage by weight of Boc-Gly-OH. Di isopropylethylamine was added at the same percentage by weight concentration asEDC and HoNb. This water coupling solution was spin coated on asubstrate to form a uniform solid layer available to couple to thesubstrate below. The wafer was then baked on a hot plate for 2 minutesat 90° C. to remove the remaining solvent (DI water) and coupled at thesame time. Next the coupling coat was washed away with DI water in astrip module.

Capping Solution was Prepared as Follows:

Acetic anhydride was obtained from Sigma Aldrich Corp. PVP was dissolvedin N methyl pyrrolidone which makes up about 1-2% by weight of the totalsolution. Next acetic anhydride was added to make up about 20-30% byweight of the solution. This capping solution was spin coated on thewafers in a capping module by spinning the wafers at 2000 rpm for 30seconds. The wafers were next baked in a cap bake module for up to 2minutes at 75° C. to complete the capping process. The remainingsolution was washed away with DI water in a strip module.

5,6 fAM Carboxy fluorescein was obtained from Anaspec. 0.1M Boc-Gly-OH(from AAPPTeC), 0.05M 5,6FAm and 0.1M HoNb (Sigma Aldrich) and 0.1M EDC(Sigma Aldrich) was dissolved in water along with 5-10% by weight Polyvinyl pyrrollidone (PolySciences). This solution is called fluoresceincoupling solution. This solution was spin coated on the wafer at 2000rpm to form a coupling dye coat. Next the wafers were baked at 65° C.for 2 mins and then the dye solution was washed away with water. Thiscompletes the coupling of fluorescein dye to measure the signals. Thesignal was then read off a fluorescence microscope. For all theexperiments, the measured signal intensity gives a direct relation ofthe coupling yield. The deprotection yield can be calculated by theamount of fluorescein coupled to the glycine on the substrate.

Comparison of Different Resists to Water Resist-1 and Water Resist-2

Water resist-1 and water resist-2 are not chemically amplified resists.These resists do not have a t-boc in the backbone and hence no chemicalamplification takes place. The post exposure bake is performed todiffuse the initial acid formed to reach the substrate. Hence thethinner the photoresist, the easier it is for diffusion of photoacid.

Exposure energy was tested in the range of 10-100 mJ/cm². Thefluorescent signal intensity shown in FIG. 11 is a relative scale from 0to 65,000.

The performance of various resist combinations was thus tested ((1 wt %pmma 2 wt % PAG 2 wt % ITX, 0.5 wt % poly 2 hydroxypropyl methacrylate 1wt % PAG 1 wt % ITX, 1 wt % pmma 2 wt % PAG 2 wt % ITX, 0.5 wt %polystyrene 1 wt % PAG, 0.5 wt % polystyrene 1 wt % PAG 1 wt % ITX, 0.5wt % pmma 0.5 wt % polystyrene 1 wt % PAG, 0.25 wt % poly ethyl acrylate0.5 wt % PAG 0.5 wt % ITX, 0.25 wt % polystyrene 0.5 wt % PAG 0.5 wt %ITX) Polymethyl methacrylate obtained from Polysciences Inc. (pmma),Photoacid generator (PAG), Isopropyl thio xanthenone obtained from SigmaAldrich (ITX)).

FIG. 11 shows the expose energy at which acid reaches the substrate foreach of the resist combinations tested. As can be seen, water resist-1and 2 each reached higher fluorescent signal intensity at lower exposeenergy as compared the other tested resists. This difference between thewater and non-water-based resists likely occurred due to the use ofpolymers in combination with the photoacid generators and thioxanthenonein the various non-water resist combinations. These polymers cannotgenerally be dissolved in water and thus a different organic solventsuch as Pgmea or ethyl alcohol is usually used. Also, the stripping ofthese resists generally involves use of organic solvents like acetonewhich would still not completely strip them off the wafer substrate andwill leave a residue. This impacts the coupling yield.

To attempt to improve the performance of some of the comparativenon-water resists, they were post baked at higher temperature of 95C and105C. This did not show any further improvement as shown in FIG. 11.

As can be seen in FIG. 11, water resist-1 and water resist-2 needs onlyapproximately 10-20 mJ/cm² of expose energy for the initial acid to beproduced, which diffuses down to reach the substrate upon post exposurebake. The lesser the expose energy required, the faster the speed ofexposure since the expose energy is directly proportional to the exposetime. This can improve throughput.

Thickness Effect on Water Resist Performance

Photoactive Formulations:

Water resist-1 was prepared by mixing the polymer polyethylene glycolmonomethyl ether (2% by weight) and polyvinyl pyrrollidone (2% byweight) in water and letting it dissolve overnight. 4Methoxyphenyl)phenyliodonium trifluoromethanesulfonate (5% by weight)was added along with isopropyl thioxanthenone (5% by weight) anddissolved overnight. Water resist-2 was prepared by mixing the polymerPoly(2-dimethylaminoethyl methacrylate) (2.5% by weight) in a solvent ofwater (90% by weight) and ethyl lactate (10% by weight) and letting itdissolve overnight. (4 methoxyphenyl)dimethylsulfonium triflate (5% byweight) was added along with isopropyl thioxanthenone and dissolvedovernight. Water resist-3 was prepared by dissolving polymerpolyethylene glycol monomethyl ether (2% by weight) and polyvinylpyrrollidone (2% by weight) in a solvent mixture comprising 50% waterand 50% ethyl lactate. 2,4-dihydroxyphenyl)dimethylsulfonium triflate(2.5% by weight) and (4 methoxyphenyl)dimethylsulfonium triflate (2.5%by weight) were added to this and were dissolved overnight. Watersoluble ITX was added at 5% by weight. Water resist-4 was prepared bydissolving polymer poly(2-dimethylaminoethyl methacrylate) (2.5% byweight) in a solvent mixture comprising 50% water and 50% ethyl lactate.2,4-dihydroxyphenyl)dimethylsulfonium triflate 2.5% and (4methoxyphenyl)dimethylsulfonium triflate (2.5% by weight) were added tothis and were dissolved overnight. Water soluble ITX was added at 5% byweight. Water resist-5 was prepared by dissolving polymerpoly(2-hydroxypropyl methacrylate) (2.5% by weight) in a solvent mixturecomprising 50% water and 50% ethyl lactate. 2,4-dihydroxyphenyldimethylsulfonium triflate (2.5% by weight) and (4methoxyphenyl)dimethylsulfonium triflate (2.5% by weight) were added tothis and were dissolved overnight. Water soluble ITX was added at 5% byweight. Water resist-6 was prepared by dissolving polymer poly 4 vinylpyridine (5% by weight) in a solvent mixture comprising 50% water and50% ethyl lactate. 2,4-dihydroxyphenyl dimethylsulfonium triflate (2.5%by weight) and (4 methoxyphenyl)dimethylsulfonium triflate (2.5% byweight) were added to this and were dissolved overnight. Water solubleITX was added at 5% by weight.

FIG. 12 shows the expose energy vs. signal/noise (SNR) of resultingchips for water resist-1 0.2 μm, water resist-2 0.15 μm, water resist-30.15 μm, water resist-4 0.1 μm, water resist-5 0.15 μm, water resist-60.1 μm which were each spin coated at 1000 rpm-2000 rpm on a spin coatmodule to the indicated thickness. FIG. 13 shows the expose energy vs.fluorescent signal of resulting chips for each of the indicated resistsand thicknesses. As can be seen in FIGS. 12-13, decreased thicknessgenerally results in a lower energy requirement to effect aciddiffusion. For example, water resist 2 with thickness 0.15 um diffusesacid at a lower energy compared to water resist 1 at 0.2 um. Thispattern was observed across water resist-3 to water resist-6.

The presence of pillars on a substrate allows for a thinner coat of thephotoresist, even as thin as 50 nm. This can lead to improvedperformance of water based photoresist.

Example 9 Production of Substrate with Carboxylic Acid Attachment Groups

This example describes how we constructed a porous layer with freecarboxylic acid groups for polypeptide synthesis/attachment. Dextran BioXtra (MW40000) is obtained from Sigma Aldrich. Bis-Polyethylene glycolcarboxy methyl ether was obtained from Sigma Aldrich. Poly vinylpyrollidone 1000000 was obtained from Poly Sciences Inc. The above threepolymers were dissolved in a solvent composition of 50% Ethyllactate/50% water by weight in a ratio of 2:2:1 by weight along with 2%by weight photoacid generator dimethyl-2,4-dihydroxyphenylsulfoniumtriflate obtained from Oakwood Chemicals Inc. This solution was spincoated onto a wafer deposited with Nickel 1000A on a Silicon substrate.The spin speed was controlled at 3000 rpm to obtain a uniform coat ofthickness 100 nm. The wafer was then exposed in a deep UV scanner NikonS 203 at 250 mJ/cm² and then baked at 65° C. for 90 sec in a hot plate.The wafer was then stripped off the coat with acetone and isopropylalcohol followed by a deionized water rinse. The substrate has a matrixof free COOH groups ready to be activated and coupled with a protein oran amino acid for peptide synthesis. The 2-dimensional concentration ofCOOH groups along the layer may be increased on a porous substrate ascompared to a planar substrate.

Dextran was coupled onto a surface derivatized wafer.1-ethyl-3-(3-dimethylaminopropyl) carbodiimide obtained from PierceScientific and N-Hydroxysuccinimide (NHS) obtained from PierceScientific were dissolved in deionized water in molar concentration of0.2M and 0.1M respectively along with 10% by weight of Dextran. Thiscoupling solution was spin coated to the wafer at a speed of 3000 rpmand baked at 65° C. for 90 sec to complete coupling of dextran-COOHsubstrate.

Bis-Polyethylene glycol carboxy methyl ether was coupled onto a surfacederivatized wafer. 1-ethyl-3-(3-dimethylaminopropyl) carbodiimideobtained from Pierce Scientific and N-Hydroxysuccinimide (NHS) obtainedfrom Pierce Scientific were dissolved in deionized water in molarconcentration of 0.2M and 0.1M respectively along with 10% by weight ofpolyethylene glycol (PEG). This coupling solution was spin coated to thewafer at a speed of 3000 rpm and baked at 65° C. for 90 sec to completecoupling of PEG-COOH substrate.

Example 10 Overlapping Peptide Array Design

In this example, a known protein (e.g., an antigen) is represented onthe array as a set of overlapping peptides. A sample obtained from asubject (in this example, serum) is assayed on the chip to identify thepeptides bound by antibodies present in the sample. The pattern ofantibody binding can be analyzed to define the epitopes recognized bythe subject's antibodies, and optionally identify the classes of thebound antibodies. See FIG. 14. In this representative example, theprotein alpha gliadin was split into overlapping peptide sequences eachhaving a length of 12 amino acids as shown in FIG. 15. The overlappingframe size shown in FIG. 15 was chosen as 2. As this example isrepresentative, only the first 29 amino acids of the entire alphagliadin protein are shown. This example is representative of arrayfeatures which comprise a plurality of distinct, nested, overlappingpeptide chains comprising subsequences derived from a source proteinhaving a known sequence.

The overlapping peptide sequences described above were then used in theconstruction of a library for a peptide array.

Example 11 Identification of a Periphilin-1 Subsequence

This example describes a method for determining the immunoactive regionsof an whole known antigen and using the immunoactive region(s) to createa set of peptides very specific to the antigen-subsequencespathologically related to a given disease; thereby reducing the numberof peptide sequences needed to represent the whole known antigen an agiven array.

Immunological binding assays were performed on an array produced usingthe materials and methods described in the Examples above. The arrayincluded the overlapping peptide sequences shown in FIG. 17. The samplesthat were used to run the assays included clinically proven celiacpositive samples.

The immunoactive regions of Periphilin-1 were determined. With referenceto FIG. 17, the microarray substrate including the Periphilin-1 sequencesplit into 12 amino acid length peptide sequences is shown. The scannedimmunoassay binding data shows the subsequence(s) that have substantiallevels of labeled antibodies bound and the subsequence(s) that do nothave substantial levels of antibodies bound. “Substantial levels” refersto binding associated with signals that are significantly abovebackground. Determination of background noise, background subtraction,and identification of signals that exceed background noise routine andwell within the level of ordinary skill in the microarray binding arts.In this example, the mean background signal intensity was estimated tobe 400 and signals were determined to be significant when they exceededbackground by a factor of 50. As the entire length of the Periphilin-1protein sequence was spanned as overlapping peptides (subsequences), thekey subsequence for antibody binding was identified as: PQQPEQII (SEQ IDNO: 23), i.e., the contiguous subsequence common to the sequences foundat array addresses 6,e through 6,g. See FIG. 17.

Example 12 Method of Identifying Epitope and Antigenic Sequences Relatedto Celiac Disorder

Determining Celiac Related Immunodominant Peptide Sequences

In this example, we determined a set of peptide sequences that interactwith immune regulatory molecules (e.g., epitopes) and are related toceliac disorder. This set of peptides was determined after two rounds ofscreening with celiac samples. The first round of screening was done bygenerating a peptide array comprising peptide subsequences tiled fromceliac related proteins. We tiled related prolamins including allgliadins, secalins, hordeins and savinas according to the schematicshown in FIG. 15 and described in Example 10 with each overlappingpeptide sequence having a length of up to 12 amino acids. The tiledsequences were also synthesized so that glutamine is replaced byglutamic acid for each possible iteration to mimic sequences present ifthe glutamine residue is deamidated to form a glutamic acid residue. Forexample, the sequence “AAIQTFQNTYQV” (SEQ ID NO: 24) was also present asa sequence where glutamine is replaced by glutamic acid in each possibleiteration, i.e., “AAIETFQNTYQV” (SEQ ID NO 25), “AAIQTFENTYQV” (SEQ IDNO 26), “AAIQTFQNTYEV” (SEQ ID NO 27), “AAIETFENTYQV” (SEQ ID NO: 28),“AAIQTFENTYEV” (SEQ ID NO: 29), “AAIETFQNTYEV” (SEQ ID NO: 30), and“AAIETFENTYEV” (SEQ ID NO: 31). An example of the deamidation (e.g., thesubstitution of glutamine with glutamic acid) of alpha gliadin peptidefragments is shown in FIG. 16.

Celiac samples were obtained from ARUP Laboratories. This sample setincluded 20 Celiac positive and 40 Celiac negative samples.

As shown in FIG. 3, our peptide arrays identified three types ofpeptides that bind to immune-related molecules associated with celiacdisorder: T peptides, B peptides, and C peptides. T peptides are thepeptides that induce energy by the immature antigen presenting cells orby stimulating the regulatory T cells. B peptides are used to diagnoseand identify the severity of the disease. C peptides are inflammationbinding peptides and are useful in determining the disease state andseverity. C peptides also increase the sensitivity of the diseasediagnosis. The method used to identify each is described in more detailbelow.

Human Leukocyte Antigen (HLA) Assay (T Peptides)

T peptides were identified from the following HLA assay for each sample.

A peptide array comprising peptide fragment sequences of relatedprolamins including all gliadins, secalins, hordeins and savinas, asdescribed above, were synthesized on the surface of the array madeaccording to methods of Example(s) 1-7. A reduction reaction wasperformed to remove the disulfide bonds that exist between cysteineresidues. This reduction reaction was not performed when epitopesinvolving cyclic peptides based on cysteine bonds are needed.Dithiothreitol was prepared in a concentration of 0.1 g per 20 mLPBS-Tween 20 (i.e., PBST). The peptide microarray (herein referred to asthe peptide chip) was immersed in the DTT solution and placed in anitrogen atmosphere at a pressure of 40 Pa at room temperature for 1hour. The peptide chip was washed by spinning it immersed in PBST for 5minutes. This procedure is repeated three times. The peptide chip wasthen washed by spinning it while immersed in methanol for 5 minutes. Thechip was then further washed while immersed in PBST buffer by spinningfor 5 minutes at 100 rpm.

The peptide fragments synthesized on the peptide chip that are veryreactive were blocked by immersing the chip in 5% bovine serum albuminin phosphate buffered saline along with superblock buffer by shaking at50 rpm for 1 hour at 37° C. The peptide chip was then incubated withceliac-positive serum and then peptides bound to HLA molecules presentin the serum were identified using a monoclonal antibody with bindingspecificity for HLA from a celiac-positive serum sample obtained fromBio-Serve. The celiac-positive serum sample was diluted to aconcentration of 1 μg/ml in PBS containing 0.2% bovine serum albumin.The peptide chip was immersed in the diluted celiac-positive serumsample and incubated at 37° C. for 24 hours without shaking. The peptidechip was then washed by shaking it for 5 minutes at 100 rpm whileimmersed in PBS with Tween-20 (i.e., PBST). This procedure was repeated3 times.

The peptide array was then analyzed to detect HLA/array peptidecomplexes using a labeled monoclonal antibody specific for HLA(monoclonal antibody obtained from Abcam). The labeled monoclonalantibody specific for the HLA was diluted in a solution of PBST tobetween 1 μg/ml and 5 μg/mL. The antibody solution was incubated withthe chip for 1 hour at 37° C. to detect to the HLA monoclonalantibody/array peptide complexes that were formed. Afterwards, the chipwas washed by immersing in PBST and spinning at 100 rpm for 5 minutes.This procedure was repeated thrice. The chip was washed by immersion indeionized water and spinning at 100 rpm for 5 minutes. This procedurewas repeated thrice. The peptide array was then analyzed to detect HLAmonoclonal antibody/array peptide complexes.

Antibody Assay (B Peptides)

B peptides were used to diagnose and identify the severity of thedisease and were identified from the following antibody assay for eachsample.

The antibody binding assay to identify B peptides was performed asfollows using materials provided in Table 9:

A peptide array comprising peptide fragment sequences synthesized on thesurface of the array was generated according to the methods describedabove. The peptide array was developed to test all 12 mer sequences fromalpha-gliadin, including sequences where glutamine was substituted withglutamate, as described above (see, e.g., FIGS. 15 and 16). A reductionreaction was performed to remove the disulfide bonds that exist betweencysteine residues. This reaction was not performed when epitopesinvolving cyclic peptides based on cysteine bonds are needed.Dithiothreitol was prepared in a concentration of 0.1 g per 20 mLPBS-Tween (PBST). The peptide microarray (herein referred to as thepeptide chip) was immersed in the DTT solution and placed in a nitrogenatmosphere at a pressure of 40 Pa at room temperature for 1 hour. Thepeptide chip was washed by spinning it immersed in PBST for 5 minutes.This procedure was repeated three times. The peptide chip was thenwashed by spinning it while immersed in methanol for 5 minutes. The chipwas then further washed while immersed in TBS buffer by spinning for 5minutes at 100 rpm.

The peptide fragments that are very reactive were blocked by immersingthe chip in 5% bovine serum albumin in phosphate buffered saline alongwith superblock buffer by shaking at 50 rpm for 1 hour at 37° C.

The chip was then incubated with a celiac positive sample. The sampledilutions varied according to the sample tested. Serum was normallydiluted 1:100 however this may vary depending on the concentration ofantibody as known to one skilled in the art. The serum was diluted in 1%BSA in solution with PBST. The chip was immersed in the diluted sampleand was incubated at 37° C. for one hour without shaking.

The chip was then washed with PBST by immersing and shaking it for 5minutes at 100 rpm. This procedure was repeated 3 times.

Secondary antibody was used to detect to the primary antibodies. Primaryantibodies include antibodies of type IgG, IgA, IgM, IgD, IgE and theirsubtypes present in sample from an individual/subject. The secondaryantibody was diluted 1:1000 in PBST and was incubated with the peptidechip for 1 hour at 37° C. in the dark. Afterwards, the chip was washedby immersion in PBST and spinning at 100 rpm for 5 minutes. Thisprocedure was repeated thrice. The chip was washed by immersion indeionized water and spinning at 100 rpm for 5 minutes. This procedurewas repeated thrice.

The peptide array was then analyzed to detect antibody/peptidecomplexes.

TABLE 9 Materials used in peptide array screening assays No. ChemicalVendor Catalogue No. 1 Superblock blocking buffer VWR PI37515 2 TBSBuffer VWR 97064-338 3 PBS with tween 20 (PBST) VWR 95059-258 4 DTTSIGMA D9779-10G ALDRICH 5 10% BSA VWR 37525, 82022-636 No. Name VendorCatalogue No. Antibodies 1 Alexa Fluor 488 Goat XMO Lifetech A11001 IgG2 Alexa Fluor 488 Goat XHU Lifetech A11013 IgG 3 Alexa Fluor 647 GoatXHU Jackson Immuno 109-606-011 IgA Research Monoclonal Antibodies 1 AntiAlpha Tubulin AALEKD ABCAM AB7291 2 Anti IL2 KPLEEVENL ABCAM AB35977 3Anti HA YPYDVEPDYA ABCAM AB130275 4 RHSVV ABCAM AB26 5 SPDDIEQWFT ABCAMAB28 6 LKWLDSFTEQ ABCAM AB1101

Celiac positive samples were assayed for antibody binding to thealpha-gliadin peptide array described earlier in this Example. Celiacpositive samples and controls were obtained from various sourcesincluding Bio-Serve and Bioreclamation.

The threshold values for negative binding, a weakly positive sequence,and a strongly positive celiac antibody binding 12-mer sequences wereestablished as follows:

<10,000=negative

10,000-20,000=weakly positive

>20,000=strongly positive

Bioinformatic Methods for Identifying Highly Informative Peptides

A set of 12-mer sequences generating weakly or strongly positive bindingsignals to celiac positive samples was compiled (see, e.g., Table A inAppendix A of U.S. Provisional Application No. 61/761,347, incorporatedherein by reference). Using a computer programmed to undertake sequenceanalysis, subsequences present within this set of 12-mer sequences weredetermined using a series of sliding windows having a minimum length of3 amino acids and maximum length of 11 amino acids. A list ofsubsequences was thus generated and stored in computer memory. Thenumber of occurrences of each subsequence in the set of 12-mer sequenceswas next determined using software that checked for the occurrence ofeach subsequence within each of the 12-mers comprising the set of 12-mersequences. The number of occurrences were tabulated, and the top 9-mostfrequently occurring subsequences were identified. We hypothesized thatthese subsequences were likely to represent highly informative sequencesthat would be useful for generating synthetic peptides that could beused to specifically identify celiac samples. For additional methodologydetails, see, e.g., Example 11 and FIG. 17).

The tabulated subsequence results obtained from IgA binding studiesusing celiac positive samples are provided, e.g., in Table B in AppendixA of U.S. Provisional Application No. 61/761,347, incorporated herein byreference. A multiplex assay was used to additionally detect bindingaffinity to IgG antibodies. The tabulated subsequence results obtainedfrom IgG binding studies using celiac positive samples are provided in,e.g., Table C in Appendix A of U.S. Provisional Application No.61/761,347, incorporated herein by reference).

It was determined that the polydiversity in the antibody is a mark ofseverity of the disease. The key immune dominant subsequence (e.g.,epitope) that simulates antibody response amongst celiac positivesubjects was found to be the peptide sequence: “QPEQPF” (SEQ ID NO: 1).However, not all celiac positive subjects showed significant binding tothis 6 mer epitope sequence. Other amino acid subsequences wereidentified that play a key role in determining the diversity of subtypesamongst patients having celiac disease.

The 9 3-mers with the highest score were combined in each possibleiteration of 2 3-mers to form 81 6 mer sequences. (FIGS. 18 and 19) withthe N-term peptides listed in the first column, and the C-term peptideslisted in the first row. The set of 6 mer sequences was used to form asecond set of sequences which are more potent than the originaldeamidated sequences and was plotted using same procedure describedabove. The sequence QPE-QPF (SEQ ID NO: 1) was found to generate samplebinding hits of 91.78% for the celiac-positive serum sample (see FIG.18, Row % QPE, Column QPF). Results are shown in FIG. 18 for IgA bindingto peptides from a celiac-positive sample, and in FIG. 19 for IgGbinding to peptides from the celiac-positive serum sample. Thesepeptides were shown to not be recognized by celiac-negative controls, orfrom positive samples from other autoimmune diseases, as describedbelow.

These sequences were compared against other diseases—40 samplesRheumatoid arthritis from Bio-Serve, 20 samples Crohns disease fromBioreclamation, 20 samples Ulcerative Colitis from Bio-Serve, 20 samplesMultiple Sclerosis from Bio-Serve, 20 samples Gastroparesis fromBio-Serve and 20 samples Systemic Lupus from Bio-Serve. None of thesesamples had antibodies against celiac sequences shown in FIGS. 18 and19.

Antibodies against specific drugs or therapeutics can be captured usingthis platform hence detecting side effects.

Cytokine Inflammation Assay (C Peptides):

Inflammation binding peptides (e.g., cytokines, TNF) are highly usefulin determining the disease state and severity but also increases thesensitivity of disease diagnosis. Another important aspect of the Cpeptides is that the response to medication can be tracked forinflammatory response. This along with the B peptides help inidentifying the refractory celiac disease. The C peptides can be used indesigning monoclonals that could serve as anti inflammatories. Cpeptides were identified from the following inflammatory assay performedon a peptide chip.

Serum from celiac samples was screened for the presence of inflammationusing the peptide chip described above. A peptide array comprisingpeptide fragment sequences synthesized on the surface of the peptidearray was generated according to the methods described above. Areduction reaction was performed to remove the disulfide bonds thatexist between cysteine residues. This reaction was not performed whenepitopes involving cyclic peptides based on cysteine bonds are needed.Dithiothreitol was prepared in a concentration of 0.1 g per 20 mLPBS-Tween (PBST). The peptide microarray (herein referred to as thepeptide chip) was immersed in the DTT solution and placed in a nitrogenatmosphere at a pressure of 40 Pa at room temperature for 1 hour. Thepeptide chip was washed by spinning it immersed in PBST for 5 minutes.This procedure was repeated three times. The peptide chip was thenwashed by spinning it while immersed in methanol for 5 minutes. The chipwas then further washed while immersed in TBS buffer by spinning for 5minutes at 100 rpm.

The peptide fragments synthesized on the peptide chip that are veryreactive were blocked by immersing the chip in 5% bovine serum albuminin phosphate buffered saline along with superblock buffer and shaking at50 rpm for 1 hour at 37° C. The peptide chip was then incubated with theserum diluted 1:100 in PBS. The peptide chip was immersed in the dilutedsample and was incubated at 37° C. for 24 hours without shaking.

The peptide chip was then washed by shaking it for 5 minutes at 100 rpmwhile immersed in PBST. This procedure was repeated 3 times.

A labeled monoclonal antibody was used to detect TNF binding. Thedilution of the monoclonal antibody can be from 1 μg/mL to 5 μg/mL. Theantibody solution was incubated with the chip for 1 hour at 37° C. todetect to the TNF/peptide complexes that were formed. Afterwards, thechip was washed by immersion in PBST and spinning at 100 rpm for 5minutes. This procedure was repeated thrice. The chip was washed byimmersion in deionized water and spinning at 100 rpm for 5 mins. Thisprocedure was repeated thrice.

The peptide array was then analyzed to detect TNF/peptide complexes. Theserum may be from a human or any animal.

Celiac Related Infections Assay:

This example provides a method of determining infections related toceliac disease patients. Antigenic peptides from bacteria, virus,fungus, parasites are tiled on the platform as 12-mer peptide sequencesand an antibody assay is performed as explained above. Table E (AppendixA) provides the list of peptides, which could provide molecular mimicryof gluten peptides, that have antibodies against common in patients withceliac disorder. An antibody assay as described in the “B peptides”assay above was performed on the peptide chip having peptides from TableE. Epitope subsequence that bind antibodies in a patient having celiacdisease are determined from the results of this assay.

Example 13

Method of Diagnosing Patients with Celiac Disorder Using a Peptide Array

Celiac Disease Detection and Subtype Diagnosis with a Single Assay:

This embodiment provides an assay protocol where in the above mentionedantibody and cytokine assays can be performed as one single assaycomprising both B peptides and C peptides identified in the assaysdescribed above. This assay protocol reduces serum usage and increasesthe sensitivity of diagnosis.

A peptide array comprising B peptide and C peptide fragment sequences(e.g., epitopes) synthesized on the surface of the peptide arrayaccording to methods 1-7 is provided. A reduction reaction is performedto remove the disulfide bonds that exist between cysteine residues. Thisreaction is not performed when epitopes involving cyclic peptides basedon cysteine bonds are needed. Dithiothreitol is prepared in aconcentration of 0.1 g per 20 mL PBS-Tween (PBST). The peptidemicroarray (herein referred to as the peptide chip) is immersed in theDTT solution and placed in a nitrogen atmosphere at a pressure of 40 Paat room temperature for 1 hour. The peptide chip is washed by spinningit immersed in PBST for 5 minutes. This procedure is repeated threetimes. The peptide chip is then washed by spinning it while immersed inmethanol for 5 minutes. The chip is then further washed while immersedin TBS buffer by spinning for 5 minutes at 100 rpm.

The peptide fragments synthesized on the peptide chip that are veryreactive are blocked by immersing the chip in 5% bovine serum albumin inphosphate buffered saline along with superblock buffer by shaking at 50rpm for 1 hour at 37° C. The peptide chip is then incubated with theserum diluted 1:100 in PBS. The peptide chip is immersed in the dilutedsample and is incubated at 37° C. for 24 hours without shaking.

The peptide chip is then washed by shaking it for 5 minutes at 100 rpmwhile immersed in PBST. This procedure is repeated 3 times.

Secondary antibody is used to detect to the primary antibodies. Primaryantibodies include antibodies of type IgG, IgA, IgM, IgD, IgE and theirsubtypes present in sample from an individual/subject. The secondaryantibody is diluted 1:1000 in PBST and is incubated with the peptidechip for 1 hour at 37° C. in the dark. Afterwards, the chip is washedimmersed in PBST by spinning at 100 rpm for 5 mins. This procedure isrepeated thrice. The chip is washed immersed in deionized water byspinning at 100 rpm for 5 mins. This procedure is repeated thrice.

The peptide array is then analyzed to detect antibody/peptide complexes.The serum may be from a human or any animal.

Example 14 Method of Treating Patients with Celiac Disorder

A mouse model is built to test the use of T peptides discovered above asa therapeutic agent. The identified T peptides are mixed in a cocktailand administered to transgenic mice. The same peptide array platformdescribed above is used to determine the cytokine profile and responseto the peptides. IFN, IL-10 and TGF-B responses are measured afteradministering the peptide cocktails. IL-10 being anti-inflammatorysuggests the presence of regulatory T cells in inducing T celltolerance. A decrease in the IFN levels suggests an immune suppressiveresponse. Hence the platform is used to identify highly specific immunesuppressive peptide cocktails.

Example 15 Lithocell Process for Generating a Peptide Array

To generate the peptide array, we used a batch processing of asemiconductor manufacturing based coater/developer module in combinationwith the lithographic deep ultraviolet tool which enabled the process tobe completely automated and hence increase throughput. The massspectrometry data obtained from this method of peptide synthesis shows asingle sharp peak as compared to the methods described in the literaturewhich show several shallow peaks.

From the PCT application, FIG. 2 A through 2 G depict the process ofmanufacturing. All these manufacturing processes were performed in acombination of lithocell and bio chemistry cell. The systems wereseparated into two or three modules as following:

Standalone—Photo-exposure tool

Standalone—Photoresist coat module

Standalone—Biochemistry track module

Any combination of above three were used to increase throughput using abatch or sequential mode processing.

This device provides a method of synthesizing biopolymers orbiomolecules in a totally automated microarray synthesis tool whichreduced the cycle time for doing one coupling reaction to less than 10minutes. The entire cycle of steps as described in FIG. 2 were splitinto a biochemistry module process and a deprotection module process(FIG. 20). A biochemistry module process involved the coupling, couplingbake, coupling strip, capping, capping bake and the capping stripprocess. A deprotection module process included photoresist coating,resist pre bake, photo exposure, post exposure bake and photo resiststrip. The time taken for a biochemistry module process was about 5minutes and the time for the deprotection module process was also 5minutes. Hence the overall time to complete one step in the sequencetook less than 10 minutes.

The embodiments of the invention include a method of synthesizingpeptide array using a microarray synthesizing cluster 1 million uniquepeptides per cm² to more than several billion unique peptides per cm²and more than 20K microarrays with 20 amino acids building block wereproduced in less than:

a. 5 days for 20mer peptide microarray in 24 hours per day synthesiswith total of 400 amino acid coupling steps.b. 10 days for 40mer peptide microarray in 24 hours per day synthesiswith total of 800-amino acids coupling steps.c. 15 days for 60mer peptides microarray in 24 hours per day synthesiswith total of 1200 amino acids coupling steps.

Referring to FIGS. 21 and 22, the wafers were cooled in a cool plate andthen coated with photoresist.

Referring to FIGS. 23, 24 and 25, the coated wafers were baked, cooled,and then moved inline to a deep ultraviolet exposure tool. (This is alsoshown in the process flow depicted in FIGS. 2A and 2B).

Referring to FIGS. 26, 27 and 28, the wafers are baked in a hot plate toenhance acid diffusion to the substrate. This does not act as a chemicalamplification step as the acid generated is not amplified upon heating.Now the wafers were cooled and rinsed with DI water to strip thephotoresist. (This is also shown in the process flow depicted in FIG.2C).

Referring to FIGS. 29 and 30, one of the twenty amino acid couplingsolutions, that were stored in Nowpak containers, were dispensedaccording to the following figures:

The following steps were followed to set up a lot of 25 wafers assuming8 ml of AA1-AA20 per wafer:

Step 1: R1 stored reagent 1—4% by weight for each EDC and HonB dissolvedin DI water. R2 stored reagent 2 DIEA. A1-A20 stored each of thenaturally occurring amino acid solution. It contained premixed solutionsof inert water soluble polymers (3% by weight PVP and 7% by weight PVA)dissolved in the ratio of 1:2.5 in deionized (DI) water which makes upabout 10% by weight of the solution along with 2% by weight amino acidconcentration. In this example, AA1 reservoir were filled with AA1 for25*8=200 ml.

Step 2: Reagent 1 was added for 0.4*20=8 ml (0.4 ml per 10 ml of A1)

Step 3: The reservoir was heated from room temperature to 60° C. andmaintained for 10 minutes starting at t₀.

Step 4: Heating was turned off and reagent 2 (0.4*20=8 mL) was addedimmediately (at t₁₀ min).

Step 5: At T₁₅ (5 minutes after reagent 2 is added and 15 minutes afterT0) the mixture was dispensed onto the first wafer.

Step 6: After all 25 wafers, the reservoir was purged with A1.

This sequence of steps complete the process for one amino acid coupling.

Referring to FIGS. 31-33, the wafers were then baked, cooled, and washedwith DI water and the entire cycle was repeated for AA2 with a differentphotomask, continuing through all peptide reservoirs to complete theentire library of peptide array.

Example 16 Pillar Substrate Preparation

Silicon wafers were obtained from University Wafers. Referring to FIG.34A, a metal was deposited on the wafers. This metal was selected fromchromium, titanium, or aluminum. The metals were deposited by a processcalled sputter deposition. Sputter deposition is a physical vapordeposition (PVD) method of depositing thin films by sputtering, that isejecting, material from a “target,” that is a source, which thendeposits onto the wafers. The thickness of metal deposition was ensuredto be at least 500 Å on top of the substrate.

Referring to FIG. 34B, silicon dioxide was deposited on the wafers. Theoxide was deposited by a process called sputter deposition. Sputterdeposition is a physical chemical vapor deposition (PECVD) method ofdepositing thin films by sputtering, that is ejecting, material from a“target,” that is a source, which then deposits onto the wafers. Thethickness of oxide deposition was ensured to be at least 500 Å on top ofthe substrate.

Referring to FIG. 34C, the first step in the preparation of a substratewas priming a starting wafer in order to promote good adhesion between aphotoactive formulation (e.g., a photoresist) and a surface. Wafercleaning was also performed, which included oxidation, oxide strip, andan ionic clean. (DI) water rinse was used to remove contaminants on thewafer surface. In wafer fabrication, silane deposition was used topromote the chemical adhesion of an organic compound (photoresist) to anon-organic substrate (wafer). The silane acts as a sort of “bridge,”with properties bind to both the photoresist and wafer surface.Typically, hexamethyldisilizane (HMDS) was used. HMDS is anorganosilicon compound that was applied on heated substrates in gaseousphase in a spray module or in liquid phase through puddle and spin in adeveloper module. This was followed by a bake step. In a puddle and spinmethod, HMDS was puddled onto the wafer for a specified time and thenwas spun and baked at temperatures of 110° C.-130° C. for 1-2 mins. In aspray module, vapors of HMDS were applied onto a heated wafer substrateat 200° C.-220° C. for 30 s-50 s.

Referring to FIG. 34C, after wafer priming, the wafers were coated witha deep ultra violet (DUV) photoresist in a photoresist coater module.Our DUV resist comprised polyhydroxystyrene-based polymers with aphotoacid generator providing the solubility change. The DUV resistfurther comprised a photosensitizer. The matrix in the polymer compriseda protecting group for e.g., tboc attached to the end group.

The DUV resist was spin coated on the wafers in a photoresist coatmodule. This module comprised a vacuum chuck held inside a cup. Thewafers were mechanically placed on the chuck by a robotic arm and thenwere spun at required speeds specified by the manufacturer to obtain theoptimum thickness.

Referring to FIG. 34C, the wafers were pre-heated in a pre-heat module.The pre-heat module included a hot plate that can be set to requiredtemperatures for the corresponding DUV resist as specified by themanufacturer. In cases for heating a batch of wafers, we used amicrowave for heating.

Referring to FIG. 34D, the wafers were exposed in a deep ultra violetradiation exposure tool through patterned photo masks.

Referring to FIG. 34E, the wafers were heated in a post exposure bakemodule. This post exposure led to chemical amplification. The resistmanufacturers provided the typical post exposure bake temperature andtime for their corresponding product. When a wafer coated with a DUVphotoresist was exposed to 248 nm light source through a reticle, aninitial photoacid or photobase was generated. The exposed portion of theresist became soluble to the developer thereby enabling patterning of0.25 micron dimensions. A post exposure bake module comprised a hotplate set to the required temperatures as specified by the manufacturer.The module comprised three vacuum pins on which the wafers were placedby a robotic arm.

Referring to FIG. 34E, the wafers were developed in a developer module.The developer module comprised a vacuum chuck that held wafers andpressurized nozzles that dispensed the developer solution on to thewafers. The dispense mode was either a puddle and spin mode or a spinand rinse mode. During the puddle and spin mode, the wafers remainedstationery on the chuck for about 30 seconds to 1 minute when thedeveloper solution was dispensed. This puddled the developer solution ontop of the wafer. After one minute, the developer solution was spunaway. During the spin and rinse mode, the developer solution wasdispensed while the wafers were spun.

Referring to FIG. 34F, the oxide was etched away in those regions thatare developed by means of a wet etch or a dry etch process. Etching is aprocess by which material is removed from the silicon substrate or fromthin films on the substrate surface. When a mask layer is used toprotect specific regions of the wafer surface, the goal of etching is toprecisely remove the material, which is not covered by the mask.Normally, etching is classified into two types: dry etching and wetetching. Wet etching uses liquid chemicals, primarily acids to etchmaterial, whereas dry etching uses gases in an excited state to etchmaterial. These processes were run to achieve an etch depth of, e.g.,500 Å.

Referring to FIG. 34G, the wafers were submerged in an oxidizer solutionovernight and then dipped in a Piranha solution for typically 1 hr.Piranha solution used was a 1:1 mixture of sulfuric acid and hydrogenperoxide. This solution was used to clean all the organic residues offthe substrates. Since the mixture is a strong oxidizer, it removed mostof the organic matter, and it hydroxylated most surfaces (i.e., add OHgroups to the surface), making the surfaces hydrophilic. This processalso included an additional step of plasma ashing.

While the invention has been particularly shown and described withreference to a preferred embodiment and various alternate embodiments,it will be understood by persons skilled in the relevant art thatvarious changes in form and details can be made therein withoutdeparting from the spirit and scope of the invention.

All references, issued patents and patent applications cited within thebody of the instant specification are hereby incorporated by referencein their entirety, for all purposes.

1. A substrate, comprising: a planar layer comprising a metal and havingan upper surface and a lower surface; and a plurality of pillarsoperatively coupled to the layer in positionally-defined locations,wherein each pillar has a planar surface extended from the layer,wherein the distance between the surface of each pillar and the uppersurface of the layer is between 1,000-5,000 angstroms, and wherein theplurality of pillars are present at a density of greater than10,000/cm².
 2. The substrate of claim 1, wherein the surface area ofeach pillar surface is at least 1 μm².
 3. The substrate of claim 1,wherein the surface area of each pillar surface has a total area of lessthan 10,000 μm².
 4. The substrate of claim 1, wherein the distancebetween the surface of each pillar and the lower surface of the layer is2,000-7,000 angstroms.
 5. The substrate of claim 1, wherein the centerof each pillar is at least 2,000 angstroms from the center of any otherpillar.
 6. The substrate of claim 1, wherein the metal is chromium,titanium, aluminum, tungsten, gold, silver, tin, lead, thallium, orindium.
 7. The substrate of claim 1, wherein each pillar comprisessilicon dioxide or silicon nitride.
 8. The substrate of claim 1, furthercomprising a linker molecule having a protecting group attached to thesurface of at least one pillar.
 9. The substrate of claim 1, furthercomprising a water soluble polymer in contact with the surface of atleast one of said pillars.
 10. The substrate of claim 1, wherein thesubstrate is coupled to a silicon wafer.
 11. The substrate of claim 1,further comprising a porous layer, said porous layer comprising aplurality of unprotected carboxylic acid side groups.
 12. The substrateof claim 11, wherein said porous layer further comprises dextran orporous silica.
 13. The substrate of claim 11, wherein said porous layerfurther comprises pores of a pore size of about 2 nm to 100 μm or aporosity of about 10-80%, and said porous layer also comprises athickness of about 0.01 μm to about 10,000 μm
 14. A method of preparinga substrate for attachment of a coupling reagent, comprising: obtaininga substrate comprising a planar layer comprising a metal and having anupper surface and a lower surface; and a plurality of pillarsoperatively coupled to the layer in positionally-defined locations,wherein each pillar has a planar surface extended from the layer,wherein the distance between the surface of each pillar and the uppersurface of the layer is between 1,000-5,000 angstroms, wherein thesurface of each pillar is parallel to the upper surface of the layer,wherein a linker molecule is attached to the surface of each pillar,wherein the substrate is contacted with a photoactive formulation, andwherein the plurality of pillars are present at a density of greaterthan 10,000/cm²; and exposing the substrate to ultraviolet light. 15.The method of claim 14, wherein the photoactive formulation comprises awater soluble photosensitizer, a water soluble photo active compound, awater soluble polymer, and a solvent, and the linker molecule comprisesa protecting group.
 16. The method of claim 14, wherein exposing thesubstrate to ultraviolet light results in removal of the protectinggroup from the linker molecule.